Secretases related to alzheimer&#39;s dementia

ABSTRACT

This invention is directed to a novel β-secretase that produces the Aβ peptide found in Alzheimer&#39;s Disease. One β-secretase is a protein having a molecular weight of about 61, 81 or 88 kDa that cleaves an amyloid precursor protein (APP) substrate. Another is a protease complex having a molecular between about 180 and 200 kDa, which, in one embodiment, contains the 61, 81, and 88 kDa proteins and, in another embodiment, contains proteins having a molecular weight of about 66, 60, 33 and 29 kDa. Another β-secretase has a molecular weight between about 50 and 90 kDA. The invention is also directed to methods of selecting agents that inhibit Aβ peptide production and treating Alzheimer&#39;s disease in patients.

[0001] This application is a continuation of application Serial No.09/294,987, filed Apr. 20, 1999, which is a continuation-in-part ofapplication Serial No. 09/173,887, filed Oct. 16, 1998, which isincorporated herein by reference.

[0002] This invention was made with government support under grantnumber NS24553 awarded by the National Institutes of NeurologicalDisease and Stroke. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to medicine. Morespecifically, the invention is directed to methods relating to treatingor preventing dementia.

[0005] 2. Background Information

[0006] Dementia is a neurological disease that results in loss ofintellectual capacity and is associated with widespread reduction in thenumber of nerve cells and brain tissue shrinkage. Memory is the mentalcapacity most often affected. The memory loss may first manifest itselfin simple absentmindedness, a tendency to forget or misplace things, orto repeat oneself in conversation. As the dementia progresses, the lossof memory broadens in scope until the patient can no longer rememberbasic social and survival skills and function independently. Dementiacan also result in a decline in the patient's language skills, spatialor temporal orientation, judgment, or other cognitive capacities.Dementia tends to run an insidious and progressive course.

[0007] Dementia results from a wide variety of distinctive pathologicalprocesses. The most common pathological process to cause dementia isAlzheimer's disease, which results in Alzheimer's-type dementia (AD).The second most common cause is multi-infarct, or vascular dementia,which results from hypertension or other vascular conditions. Dementiacan also result from infectious disease, such as in Creutzfeldt-Jakobdisease. Dementia occurs in Huntington's disease, which is caused by anautosomal dominant gene mutation, and in Parkinson's disease, which isassociated with a motor disorder. Dementia also occurs from head injuryand tumors.

[0008] Rare before age 50, AD affects nearly half of all people past theage of 85, which is the most rapidly growing portion of the UnitedStates population. As such, the current 4 million AD patients in theUnited States are expected to increase to about 14 million by the middleof the next century.

[0009] No method of preventing AD is known and treatment is primarilysupportive, such as that provided by a family member in attendance.Stimulated memory exercises on a regular basis have been shown to slow,but not stop, memory loss. A few drugs, such as tacrine, result in amodest temporary improvement of cognition but do not stop theprogression of dementia.

[0010] A hallmark of AD is the accumulation in brain of extracellularinsoluble deposits called amyloid plaques, and abnormal lesions withinneuronal cells called neurofibrillary tangles. The presence of amyloidplaques, together with neurofibrillary tangles, are the basis fordefinitive pathological diagnosis of AD. Increased plaque formation isassociated with increased risk of AD.

[0011] The major components of amyloid plaques are the amyloidβ-peptides, also called Aβ peptides, which consist of three proteinshaving 40, 42 or 43 amino acids, designated as the Aβ₁₋₄₀, Aβ₁₋₄₂, andAβ₁₋₄₃ peptides. The amino acid sequences of the Aβ peptides are knownand the sequence of the Aβ₁₋₄₂ is identical to that of the Aβ₁₋₄₀peptide, except that the Aβ₁₋₄₂ peptide contains two additional aminoacids at its carboxyl (COOH) terminus. Similarly, the amino acidsequence of the Aβ₁₋₄₃ peptide is identical to that of the Aβ₁₋₄₂peptide except that the Aβ₁₋₄₃ peptide contains one additional aminoacid at its carboxyl terminus. The Aβ peptides are thought to cause thenerve cell destruction in AD, in part, because they are toxic to neuronsin vitro and in vivo.

[0012] The Aβ peptides are derived from larger amyloid precursorproteins (APP proteins), which consist of four proteins, designated asthe APP₆₉₅, APP₇₁₄, APP₇₅₁, and APP₇₇₁ proteins, which contain 695, 714,751 or 771 amino acids, respectively. The different APP proteins resultfrom alternative ribonucleic acid splicing of a single APP gene product.The amino acid sequences of the APP proteins are also known and each APPprotein contains the amino acid sequences of the Aβ peptides.

[0013] Proteases are believed to produce the Aβ peptides by recognizingand cleaving specific amino acid sequences within the APP proteins at ornear the ends of the Aβ peptides. Such sequence specific proteases arethought to exist because they are necessary to produce from the APPproteins the Aβ₁₋₄₀, Aβ₁₋₄₂, and Aβ₁₋₄₃ peptides consistently found inplaques.

[0014] But the proteases have not been isolated. Nonetheless, they havebeen named “secretases” because the Aβ peptides which they produce aresecreted by cells into the extracellular environment. Moreover, thesecretases have been named according to the cleavages that must occur toproduce the Aβ peptides. The secretase that cleaves the amino terminalend of the Aβ peptides is called the β-secretase and that which cleavesthe carboxyl terminal end of the Aβ peptides is called the γ-secretase.The γ-secretase determines whether the Aβ₁₋₄₀, Aβ₁₋₄₂, or Aβ₁₋₄₃ peptideis produced (see FIG. 1). But since the secretases have not beenisolated, the terms β-secretase and γ-secretase each could relate to oneor several protease species.

[0015] In addition to the Aβ peptides, proteolytic cleavage of anotherspecific amino acid sequence within the APP proteins is known to occurand to produce α-APP and 10 kilodalton (kDa) fragments. That amino acidsequence lies within the Aβ peptide amino acid sequence of the APPproteins. Like the β-secretase and the γ-secretase, the proteaseresponsible for that cleavage has also not been isolated but has beennamed the α-secretase (see FIG. 1). Significantly, the products producedby the α-secretase cleavage, the α-APP and the 10 kilodalton (kDa)fragments, do not form senile plaques.

[0016] Proteases can be isolated from tissue homogenates or lysed cellsamples, but those samples can contain the proteases from cellorganelles in which the product is not produced, but which may be ableto cleave in vitro the precursor protein to produce the product. Thus, aproblem in using such samples to isolate the secretases has been thatproteases which produce the Aβ peptide in vitro, but not in vivo, may beerroneously isolated.

[0017] The problem can be avoided by isolating the secretase from cellorganelles in which the APP proteins are processed in vivo. A cellorganelle thought to be a site in which such processing occurs is thesecretory vesicles of brain neuronal cells. But methods have not beendeveloped to obtain sufficient amounts of pure secretory vesicles fromneuronal cells to assay for secretase activity in those vesicles.

[0018] Large amounts of pure secretory vesicles can be obtained fromchromaffin cells, neuroendocrine cells of the adrenal medulla, and havebeen used to obtain proteases. For example, carboxypeptidase H (CPH),prohormone thiol protease (PTP), and the prohormone convertases (PC1 andPC2), which process precursor proteins into peptides having opiateactivity have been obtained from such vesicles. But chromaffin cellshave not been shown to produce the Aβ peptides or have secretaseactivity.

[0019] The β-secretase, γ-secretase, and α-secretase must be isolated tounderstand how the neurotoxic Aβ peptides are produced so that AD can beprevented or treated. To isolate the secretase, new methods are neededfor assaying the proteolytic activity of secretases in substantiallypurified preparations of the cell organelles in which the APP protein isprocessed in vivo. Moreover, new screening methods for selecting agentsthat affect the proteolytic activity of the secretases are needed todevelop new pharmaceuticals for treating or preventing AD. Further, suchnew methods need to be applied and the secretases isolated.

[0020] The invention satisfies these needs by providing new methods ofdetermining the proteolytic activity of secretases and isolatingsecretases having that activity. The invention also provides newscreening methods for selecting agents that affect the activity of suchsecretases. Moreover, the invention discloses novel β-secretasesobtained by such methods as well as methods of selecting agentsinhibiting production of Aβ peptides by inhibiting the activity of thoseβ-secretases.

SUMMARY OF THE INVENTION

[0021] The invention is directed to various novel β-secretases. One suchβ-secretase contains a protein having a molecular weight of about 61, 81or 88 kDa as determined by cleavage of an APP substrate in anon-reducing SDS-PAGE in gel activity assay. In one embodiment, theβ-secretase contains a protein that cleaves the APP substrate in theβ-secretase recognition site at the Lys-Met bond.

[0022] Another is a protease complex having a molecular weight betweenabout 180 and 200 kiloDaltons (kDa) as determined by Sephacrylchromatography that cleaves an APP substrate. In one embodiment, theprotease complex cleaves the APP substrate in the β-secretaserecognition site at the Lys-Met bond. In another embodiment, theprotease complex contains proteins having molecular weights of about 66,60, 33 and 29 kDa as determined by a reducing SDS-PAGE in gel proteinstaining assay. In another embodiment, the protease complex containisproteins having molecular weights of about 61, 81 and 88 kDa asdetermined by cleavage of an APP substrate in a non-reducing SDS-PAGE ingel activity assay.

[0023] Another β-secretase has a molecular weight between about 50 and90 kDA as determined by Sephacryl chromatography and cleaves an APPsubstrate. In one embodiment, that β-secretase cleaves the APP substratein the β-secretase recognition site at the Met-Asp bond. In anotherembodiment, the β-secretase contains 2 proteins having differentelectronegative charges as determined by ion exchange chromatography.

[0024] The invention is also directed to a method of selecting an agentthat inhibits cleavage of the APP substrate by the β-secretasesdescribed above. The invention is further directed to a method ofinhibiting production of an Aβ peptide by a cell or by an Alzheimer'sdisease patient using such a selected agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1. The upper bar is a diagram of an amyloid precursor protein(APP protein). The amino and carboxyl termini of the APP protein areindicated by the letters “N” and “C,” respectively. The relativelocation of various known regions within the APP protein are indicated,including the signal peptide (SP), cysteine-rich (C-rich), negativelycharged ((−)charged), protease inhibitor, Ox antigen (Ox),transmembrane, cytoplasmic and Aβ peptide regions. The amino acidsequence of the Aβ peptides and regions flanking the Aβ peptides isshown by the letters below the amyloid precursor protein (SEQ ID NO.:1).Each letter represents an amino acid according to the conventionalsingle letter amino acid abbreviation format. Scissile bonds within theamino acid sequence cleaved by the β-, γ-, or α-secretases are indicatedby the β, γ, and α labels. Three scissile bonds cleaved by β-secretaseswhich, in combination with scissile bond cleaved by the γ-secretase,produce the Aβ₁₋₄₀, Aβ₁₋₄₂, or Aβ₁₋₄₃ peptide. The three parallel linesbelow the amino acid sequence identify the amino acid sequences of theAβ₁₋₄₀, Aβ₁₋₄₂, and Aβ₁₋₄₃ peptides.

[0026]FIG. 2. The bonds, labeled #1, #2, and #3, in theZ*Val-Lys-Met-MCA substrate cleaved by a secretase having endoproteaseactivity are shown. The Z, Val, Lys, Met, and MCA in the substraterepresent a carbobenzoxy, valine, lysine, methionine, andaminomethylcourmarinamide group, respectively. The star (*) and dash (-)represent nonpeptide and peptide bonds, respectively

[0027]FIG. 3. The fluorescence activity is plotted as a function of thepH at which a lysate of substantially pure chromaffin vesicles isincubated with the Z*Val-Lys-Met-MCA substrate. The fluorescenceactivity is the relative fluorescence of the free MCA (AMC) released byproteolytic cleavage of the substrate.

[0028]FIG. 4. The fluorescence activity is plotted as a function of thepH at which a lysate of substantially pure chromaffin vesicles isincubated with the Met-MCA substrate. The fluorescence activity is therelative fluorescence of the free MCA (AMC) released by proteolyticcleavage of the substrate.

[0029]FIG. 5. The fluorescence activity is plotted as a function of thepH at which the lysate of substantially pure chromaffin vesicles isincubated with the Lys-MCA substrate. The fluorescence activity is therelative fluorescence of the free MCA (AMC) released by proteolyticcleavage of the substrate.

[0030]FIG. 6. The fluorescence activity is plotted as a function of thepH at which the lysate of substantially pure chromaffin vesicles isincubated with the Z*Val-Lys-Met-MCA substrate in the presence andabsence of DTT (closed and open squares, respectively). The fluorescenceactivity is the relative fluorescence of the free MCA (AMC) released byproteolytic cleavage of the substrate.

[0031]FIG. 7. The fluorescence activity is plotted as a function of thepH at which the lysate of substantially pure chromaffin vesicles isincubated with the Z*Val-Lys-Met-MCA substrate in the presence of DTTwithout aminopeptidase M (open triangles), with basic pH buffer (opensquares), or with aminopeptidase M (closed squares). The fluorescenceactivity is the relative fluorescence of the free MCA (AMC) released byproteolytic cleavage of the substrate.

[0032]FIGS. 8. The isolation procedure used to obtain Peak I and Peak IIis diagramed.

[0033]FIG. 9. The fluorescence activity is plotted as a function of thefraction number (#) obtained from the Sephacryl S200 in the procedurediagramed in FIG. 8. Fraction numbers 30 to 40, and 40 to 50 containPeak I and Peak II, respectively. The activity is that which resultsfrom cleavage of the Z*Val-Lys-Met-MCA substrate by the fraction withoutaminopeptidase M (open squares), or with aminopeptidase M (closedsquares). The fluorescence activity is in pmol of free MCA permicroliter (AMC/μl). The γ-globulin, BSA, and myoglobin are calibrationweight standards.

[0034]FIG. 10. The procedure used to isolate the β-secretases from PeakI is diagramed.

[0035]FIG. 11. The procedure sued to isolate the β-secretases from PeakII is diagramed.

DETAILED DESCRIPTION OF THE INVENTION

[0036] This invention provides an assay for the proteolytic activity ofsecretases, particularly the β-secretase and the γ-secretase thatproduce the Aβ peptides found in the plaques of AD patients. The methodis novel because the activity is detected in a substantially purifiedpreparation of vesicles in which APP protein processing occurs in vivo.Based on that activity assay, new methods are disclosed to isolate thesecretases from such substantially purified preparations. Isolating thesecretases from the cell organelles in which the APP protein isprocessed insures that the secretases are the in vivo secretases and notmerely a protease from a cell organelle in which such processing doesnot occur, but which is capable of cleaving the APP protein in vitro.The invention further provides methods of selecting an agent thataffects the proteolytic activity of the substantially purified vesicles,the isolated secretase, or the cells containing the vesicles.

[0037] As discussed in Examples V and VII below, the secretory vesiclesof chromaffin cells of the adrenal medulla, herein called “chromaffinvesicles,” were discovered to contain Aβ peptides, specifically theAβ₁₋₄₀ and the AB₁₄₂ peptides, and that chromaffin cells can secretethese peptides. As such, the chromaffin vesicles were found to containthe in vivo product produced by APP protein processing. Moreover, thevesicles were known to contain the APP proteins and presenilin 1protein, a protein that affects secretase activity (see Vassilacopoulouet al., J. Neurochem. 64:2140-2146, (1995); Tezapsidis et al., Biochem.37(5):1274-1282, (1998); Borchelt et al., Neuron 17:1005-1013, (1996);St. George-Hyslop et al., Science 264:1336-1340, (1994); Alzheimer'sDisease Collaborative Group, Nature Genet. 11:219-222, (1995); and Wascoet al., Nature Med. 1:848, (1995)).

[0038] Chromaffin vesicles can be obtained in relatively largequantities. That capability, combined with the discovery that thechromaffin vesicles contained the Aβ peptides, permitted for the firsttime assaying a substantially pure preparation of cell organelles inwhich APP processing occurs for the proteolytic activity of a secretase.Further, chromaffin vesicles can be obtained in amounts which alsopermit isolating and sequencing the secretases present in those cellorganelles.

[0039] As described more fully below in Examples I through XV, bovinechromaffin vesicles were initially discovered to have secretaseproteolytic activity. Moreover, it was found that secretases having thatactivity could be isolated from bovine chromaffin vesicles. But the samemethods can be applied to other mammalian species, including humans. Assuch, secretases from various mammalian species can be assayed for andisolated using the methods disclosed herein.

[0040] Further, the amino acid sequence of a bovine secretase is likelyto be highly homologous with that of the corresponding human secretasebecause other bovine proteases are known to have a high degree ofhomology with the corresponding human protease. For example, the aminoacid sequence of the bovine carboxypeptidase H is about 96% homologouswith the corresponding human carboxypeptidase H (Hook et al., Nature,295:341-342, (1982); Fricker et al., Nature, 323:461-464, (1986); andManser et al., Biochem. J., 267:517-525, (1990)). Once the amino acidsequence of a secretase from one species is obtained, the correspondingsecretase in other species thus can be obtained using recombinantmethods such as those described below.

[0041] The term “secretase” as used herein means a protease that cleavesan APP protein in vivo. A protease is a protein that enzymaticallybreaks a peptide bond between two amino acids or an amino acid andchemical moiety as described below. Although the term secretase impliesthe production of a soluble, secreted peptide, an APP derived productproduced by a secretase of the invention need not necessarily be solubleor secreted. “Secretase” includes those secretases referred to asβ-secretase and γ-secretase, each of which can relate to one or moreprotease species that produce the Aβ peptides. “Secretase” also includesthe secretase referred to as α-secretase which can relate to one or moreprotease species that produce the α-APP fragment or the 10 kDa fragment.

[0042] The term “vesicles” as used herein refers to secretory vesiclesand condensing vacuoles of the secretory pathway. Such vesicles have amembrane that forms a spherical shaped structure and that separates thecontents of the vesicles from the rest of the cell. The vesicles processand store their contents until such time as the contents are secretedinto the extracellular environment by a cellular process calledexocytosis, which occurs by fusion of the secretory vesicle membranewith the cell membrane. The secretion can occur in response to atriggering event in the cell such as a hormone binding to a receptor.Vesicles can be identified by their characteristic morphology or by thepresence of a chemical compound characteristic of such vesicles.

[0043] As used herein, the term “substantially pure” as used in regardto vesicles means that at least about 80% of the cell organelles in asample are vesicles. Usually a substantially pure sample has about 95%or more vesicles and often has about 99% or more vesicles. Substantiallypure vesicles include a single isolated vesicle. Substantially purechromaffin vesicles result after approximately an 8-fold purificationfrom the cell homogenate as described below in Example II.

Methods of Determining the Proteolytic Activity of a Secretase

[0044] One aspect of the invention is an assay for determining theproteolytic activity of a secretase by obtaining substantially purevesicles, permeabilizing the vesicles, and incubating the permeablizedvesicles with an APP substrate in conditions which allow the secretaseto cleave the APP substrate. The cleavage of the APP substrate isdetected and the activity of the secretase is thereby determined.

[0045] The vesicles can be obtained from any cell that contains vesiclesin which APP protein processing occurs. Vesicles in which suchprocessing occurs can be assayed for by the presence of an Aβ peptide,an α-APP fragment or a 10 kDa fragment in the vesicles using methodsdescribed below. Cells containing such vesicles include, for example,neuronal cells from brain tissue, chromaffin cells from adrenal medullatissue, and platelets from blood. Tissue samples containing such cellscan be surgically removed or platelets can be isolated from blood bymeans known in the art. For tissue samples, the vesicles can be obtainedfrom mechanically homogenized tissue or from tissue disassociated byincubation with collagenase and DNAse (see, for example, Krieger et al.,Biochemistry, 31, 4223-4231, (1992); Hook et al., J. Biol. Chem.,260:5991-5997, (1985); and Tezapsidis et al., J. Biol. Chem.,270:13285-13290, (1995), which are incorporated herein by reference).

[0046] The substantially pure vesicles can be obtained from the tissuehomogenates or lysed cells using known methods (see Current Protocols inProtein Science, Vol. 1 and 2, Coligan et al., Ed., John Wiley and Sons,Pub., Chapter 4, pp. 4.0.1-4.3.21, (1997)). For example, substantiallypure secretory vesicles can be isolated using discontinuous sucrosegradient centrifugation methods (see Krieger et al., ibid.; andYasothornsrikul et al., J. Neurochem. 70, 153-163, (1998)). Vesiclesalso can be isolated using metrizamide gradient centrifugation (Toominet al., Biochem. Biophys. Res. Commun., 183:449-455, (1992); and Loh etal., J. Biol. Chem., 259:8238-8245, (1984), or percoll gradientcentrifugation (Russell, Anal. Biochem., 113:229-238, (1981). Ifdesired, capillary electrophoresis methods can be used to isolateindividual vesicles (Chie et al, Science, 279:1190-1193, (1998)). Othermethods, including differential centrifugation, fluorescence-activatedsorting of organelles, immunoabsorption isolation, elutriationcentrifugation, gel filtration, magnetic affinity chromatography,protein chromatographic resins, agarose gel electrophoresis, and freeflow electrophoresis methods, also can be used to obtain substantiallypure vesicles. The references cited in this paragraph are incorporatedby reference.

[0047] The purity of the secretory vesicle preparation can be assayedfor by morphological or chemical means. For example, vesicles can beidentified by their characteristic morphology as observed by electronmicroscopy. The vesicles can be prepared for electron microscopy usingvarious methods including ultra-thin sectioning and freeze-fracturemethods. Vesicles also can be identified by the presence of acharacteristic neurotransmitter or hormone present in such vesicles suchas the (Met)enkephalin, catecholamines, chromogranins, neuropeptide Y,vasoactive intestinal peptide, somatostatin, and galanin found inchromaffin vesicles (Hook and Eiden, FEBS Lett. 172:212-218, (1984); Lohet al., J. Biol. Chem. 259:8238-8245, (1984); Yasothornsrikul et al., J.Neurochem. 70:153-163, (1998), which are incorporated herein byreference). The presence of the characteristic chemical compound can bedetermined by various means including, for example, by radioactive,fluorescent, cytochemical, immunological assays, or mass spectrometrymethods. More specifically, such assays include radioimmunoassays,western blots or MALDI mass spectrometry. In addition, vesicles can beassayed using light and electron microscopic methods, fluorescent cellactivated cell sorter methods, density gradient fractionation methods,immunoabsorption methods, or biochemical methods.

[0048] The activity of the secretases can be preserved while thevesicles are purified using known methods. For example, the vesicles canbe obtained at a low temperature (e.g. 4° C.) and frozen (e.g. −70° C.)prior to assaying for secretase activity. The activity can also bepreserved by obtaining the vesicles in the presence of a stabilizingagent known to preserve protease activity (see Enzymes, Dixon et al.,Eds., Academic Press, Pub., pp. 11-12, (1979), and Current Protocols inProtein Science, , Vol. 1 and 2, Coligan et al., Ed., John Wiley andSons, Pub., Chapter 4, pp. 4.5.1-4.5.36, (1997), which are incorporatedherein by reference). Known stabilizing agents include proteins,detergents and salts, such as albumin protein, CHAPS, EDTA, glycerol,and NaCl. Reducing agents are also known to preserve protein functionand can be used (see Voet et al., Biochemistry, John Wiley and Sons,Pub., pp. 382-388 and 750-755, (1990), which is incorporated herein byreference). Known reducing agents include, for example,β-mercaptoethanol, DTT, and reduced glutathione (see Example VIII).

[0049] So that secretases within the vesicles are accessible to an APPsubstrate in an incubation solution, the vesicles are permeablized (seeVoet et al., Biochemistry, John Wiley and Sons, Pub., pp. 284-288,(1990); and Krieger et al., ibid., which are incorporated herein byreference). Permeabilizing can result in a continuum of affects on thevesicle ranging from the formation of one or more holes in the membraneto complete lysis of the membrane. Vesicles can be permeablized, forexample, by contact with a detergent or a disruptive agent such asCHAPS, sodium dodecyl sulfate, sodium cholate, digitonin, Brij 30 orTRITON X-100. Vesicles can be lysed, for example, by freeze-thawing,especially in a potassium chloride solution, by suspension in ahypoosmotic solution or by mechanical means such as sonication.

[0050] The permeablized vesicles are incubated with an APP substrateunder appropriate conditions for cleavage of the APP substrate by asecretase. Various incubation conditions are known to affect proteasecleavage. For example, the pH of the interior of chromaffin vesicles isacidic and some proteases in those vesicles are known to only functionin an acidic incubation solution (Pollard et al., J. Biol. Chem.254:1170-1177, (1979); and Hook et al., FASEB J. 8:1269-1278, (1994)).Thus, a condition for cleavage of the APP substrate includes anincubation solution having a pH of about 7.0 or less. But secretases invesicles are released by cells into the extracellular environment, whichcan have a neutral or basic pH. Thus, vesicles can contain secretasesthat function at the neutral or basic pH of the extracellularenvironment and, as such, that pH can also be an appropriate condition.The pH of the incubation solution can be adjusted using known buffers(see Voet et al., Biochemistry, John Wiley and Sons, Pub., pp. 35-39,(1990)). Such buffers include, for example, citric acid, sodiumphosphate, MES, HEPES and Tris-HCl buffers. The pH of the incubationsolution can be determined using known methods such as, pH colorindicators in liquid or paper formats, or pH meters. Examples III, IV,VIII, and IX show that the pH of the incubation solution can affect theactivity of secretases.

[0051] Other conditions that affect the cleavage include the incubationtemperature and incubation time. Proteolytic activity is a function oftemperature with excessively low or high temperatures resulting in nodetectable activity. An incubation temperature thus is any temperaturewhich allows detection of a cleaved APP substrate. Usually an incubationtemperature of about 30° to 45° C., with a typical temperature of about35° to 40° C., and often a temperature of about 37° C. is used. Althoughnot required, a constant temperature during the incubation time ispreferred and can be achieved using an incubator, water bath or otherknown means. An insufficient or excessive incubation time results in toolittle production or too much degradation of the product to be detected.The incubation time for cleavage of an APP substrate is that amount oftime which allows cleavage of the APP substrate to be detected. Apreferred incubation time allows the cleavage of an APP substrate to goto completion, for example, in about 2 to 8 hours.

[0052] The proteolytic activity of a secretase is determined by thecleavage of an APP substrate. An “APP substrate” as used herein is acompound having a stereochemical structure that is the same as, or amimic of, an amino acid sequence in an APP protein, an Aβ peptide, anα-APP fragment or a 10 kDa fragment recognized by a secretase. Thus, anAPP substrate for detecting a β- or γ-secretase includes, for example,the APP₆₉₅, APP₇₁₄, APP₇₅₁, and APP₇₇₁ proteins and an APP substrate fordetecting an α-secretase includes, for example, those proteins and theAβ peptides. As discussed above, such proteins, peptides and fragmentshave been isolated and characterized (Kang et al., Nature 325:733-736,(1987); Kitaguchi et al., Nature 331:530-532, (1988); Ponte et al.,Nature 331:525-527, (1988); Tanzi et al., Nature 331, 528-530, (1988);Tanzi et al., Science 235:880-884, (1987), Glenner et al., Biochem.Biophys. Res. Commun. 120, 885-890, (1984); Masters et al., Proc. Natl.Acad. Sci. USA 82: 4245-4249, (1985); Selkoe et al., J. Neurochem. 146:1820-1834, (1986); Selkoe, J. Biol. Chem. 271:18295-18298, (1996); Mannet al., Amer. J. Pathology 148: 1257-66, (1996); Masters et al., Proc.Natl. Acad. Sci. USA 82: 4245-4249, (1985); Selkoe et al., J. Neurochem.146: 1820-1834, (1986); Selkoe, J. Biol. Chem. 271:18295-18298, (1996);and Mann et al., Amer. J. Pathology 148: 1257-66, (1996)).

[0053] Such APP substrates can be produced by various methods known inthe art (Knops et al., J. Biol. Chem. 266:7285-7290, (1991); Hines etal., Cell. Molec. Biol. Res. 40:273-284, (1994)). For example, the APPproteins can be made using recombinant technology and cloning the cDNAthat encodes the proteins into a suitable expression system. An APPprotein cDNA can be obtained, for example, by screening a human braincDNA library with a DNA probe consisting of an oligonucleotidecomplementary to the APP protein cDNA, a PCR-generated DNA fragment ofthe APP protein cDNA, or a DNA fragment of the APP protein cDNA from anexpressed sequence tagged (EST) database. Expression systems to produceAPP proteins include, for example, E. coli., baculovirus-infected insectcells, yeast cells, and mammalian cells. Alternatively, such proteinscan be produced using in vitro methods, which transcribe and translatethe RNA that encodes these proteins to produce the proteins. An APP soproduced can be purified using methods such as described herein orotherwise known in the art.

[0054] An APP substrate is also an APP substrate-fusion substrate, inwhich a protein or peptide is attached to an APP substrate for thepurpose of facilitating the isolation of the APP substrate. Proteins orpolypeptides that facilitate purification include, for example,maltose-binding protein and multi-histidine polypeptides attached to theamino or carboxyl terminal end of the APP substrate. Thus, an example ofan APP-fusion substrate is a multi-histidine polypeptide attached to thecarboxyl terminus of an APP₆₉₅, APP₇₁₄, APP₇₅₁, or APP₇₇₁ protein. SuchAPP-fusion substrates can be produced using known methods such as byexpression of the cDNA that encodes the APP-fusion substrate in asuitable expression system or in vitro translation of the encoding RNA.The APP-fusion substrates so produced can be purified by affinitybinding to a column, such as by amylose, nickel or anti-APP antibodycolumn chromatography.

[0055] Peptides are also known to function as protease substrates (seeSarath et al., Protease assay methods, In: Proteolytic Enzymes, APractical Approach, R. J. Beynon and J. S. Bond, Eds., Oxford UniversityPress, Pub., Chapter 3, pp 25-55, (1989). Often such a peptide substratewill contain the amino acids at a scissile bond in a precursor protein(see Benyon et al., The Schecter and Berger Nomenclature for ProteaseSubstrates, In: Proteolytic Enzymes, A Practical Approach, R. J. Beynonand J. S. Bond, Eds., Oxford University Press, Pub., especially,Appendix 1, pp 231, (1989); and Barrett, An Introduction to theProteinases, In: Proteinase Inhibitors, A. J. Barrett and G. Salvesen,Eds., Elsevier, Pub., Chapter 1, pp. 3-18, (1986)). A scissile bond isthe peptide bond cleaved by a protease in a precursor protein. The aminoacid on the amino terminal side of the scissile bond is often called theP1 amino acid and that on the carboxyl terminal side the P1′ amino acid.

[0056] A protease that cleaves a scissile bond binds the P1 and P1′amino acids. For some proteases, the P1 amino acid is the primarydeterminant for protease binding to the precursor protein. For example,the protease trypsin is known to have a marked preference for bindingbasic P1 amino acids. Peptide substrates often contain the amino acidsattached to the amino terminal side of a P1 amino acid because thoseamino acids can influence the determinant effect of the P1 amino acid.

[0057] An APP substrate also includes a peptide having an amino acidsequence recognized by a secretase containing a P1 or P1′ amino acid, orboth, of a scissile bond in an APP protein and one or more of the aminoacids in the APP protein adjacent to either the P1 or P1′ amino acids orboth. For example, as shown in FIG. 1, a β-secretase scissile bond isbetween the P1 amino acid methionine (Met or M) and the P1′ amino acidaspartic acid (Asp or A). A β-secretase recognition site thus includes,for example, a Met-Asp substrate.

[0058] Often an APP substrate is a peptide containing the P1 and P1′amino acids of a scissile bond in an APP protein and the one or twoamino acids in the APP protein attached to the amino terminal side ofthe P1 amino acid. For example, as shown in FIG. 1, a lysine (Lys or K)is attached to the amino terminal side of the PI amino acid of theβ-secretase scissile bond and a valine (Val or V) is attached to theamino terminal side of the Lys. Thus, an APP substrate for theβ-secretase includes the Lys-Met-Asp and Val-Lys-Met-Asp (SEQ. ID NO.:1)substrates.

[0059] The APP substrate peptide containing the P1 and P1′ amino acidsof a scissile bond in an APP protein can be determined for theγ-secretase and the α-secretase in the same manner. For example, asshown in FIG. 1, the γ-secretase scissile bond of the Aβ₁₋₄₀ peptide hasa Val P1 amino acid, an isoleucine (Ile or I) P1′ amino acid, a secondVal attached to the amino terminal side of the P1 amino acid and aglycine (Gly or G) attached to the amino terminal side of the secondVal. As such, the γ-secretase recognition site for the Aβ₁₋₄₀ peptideincludes, for example, the Val-Ile, Val-Val-Ile and Gly-Val-Val-Ile (SEQID NO.:2) substrates. The γ-secretase recognition site for the Aβ₁₋₄₂peptide thus includes, for example, the Ala-Thr, Ile-Ala-Thr andVal-Ile-Ala-Thr (SEQ ID NO.:3) substrates and that the g-secretaserecognition site for the Aβ₁₋₄₃ peptide includes, for example, theThr-Val, Ala-Thr-Val, and Ile-Ala-Thr-Val (SEQ ID NO.:4) sequences.Similarly, the α-secretase recognition site can be determined from theamino acids in the APP protein surrounding the α-secretase scissilebond.

[0060] Proteases are known to have endoprotease, aminopeptidase, orcarboxypeptidase activity, or a combination of these activities (seeSarath et al., ibid.) . A protease having endoprotease activity cleavesthe peptide bond between two adjacent amino acids, neither of which is aterminal amino acid, or, as discussed below. between a non-terminalamino acid and a terminal blocking group. A protease havingaminopeptidase activity only cleaves the peptide bond between the aminoterminal amino acid and its adjacent amino acid. A protease havingcarboxypeptidase activity only cleaves the peptide bond between thecarboxyl terminal amino acid and its adjacent amino acid.

[0061] Secretases of the invention also can have endoprotease,aminopeptidase, or carboxypeptidase activity, or a combination of theseactivities. For example, an Aβ peptide can be cleaved from an APPprotein directly by endoprotease cleavage of the scissile bonds at bothends of the Aβ peptide. But an Aβ peptide also can be produced by anendoprotease cleavage of a scissile bond distal to the terminal aminoacids of the Aβ peptide followed by aminopeptidase or carboxypeptidasecleavage of the amino acids flanking the terminal amino acids of the Aβpeptide.

[0062] An APP substrate often contains one or more amino terminal orcarboxyl terminal blocking groups, which prevent aminopeptidase orcarboxypeptidase cleavage, respectively (see Sarath et al., ibid.). Butan amino terminal blocking group does not prevent carboxypeptidase and,conversely, a carboxyl terminal blocking group does not preventaminopeptidase cleavage. As such, an APP substrate can often containboth an amino terminal and carboxy terminal blocking group to preventboth aminopeptidase and carboxylpeptidase cleavage. An APP substratecontaining both blocking groups can only be cleaved, if at all, by asecretase having endoprotease activity.

[0063] Blocking groups and methods of making substrates containingblocking groups are known in the art (see, for example, Methods inEnzymology, Vol. 244, “Proteolytic Enzymes,” A. J. Barrett, Ed.,Chapters 46, 47, and 48, (1994); and Green and Wuts, Protective Groupsin Organic Synthesis, John Wiley and Sons, Pub., (1991) which are hereinincorporated by reference). Amino terminal blocking groups include, forexample, acyl (Ac), benzoyl (Bz), succinyl (Suc), carbobenzoxy (Z),p-bromocarbobenzoxy, p-chlorocarbobenzoxy, p-methoxycarbobenzoxy,p-methoxyphenylazocarbobenzoxy, p-nitrocarbobenzoxy,p-phenylazocarbobenoxy, tert-butoxycarbonyl (Boc), benzoyl and the like.Carboxyl blocking groups include, for example, aminomethylcourmarinamide(MCA), the diazomethanes, the p-nitroanlide (pNA), pNA•Tosylate,2-naphthylamine, the acyloxymethanes, including the(benzoyloxy)methanes, (alkyloxy)methanes, the N,O-diacyl hydroxamates,including the N-aminoacyl-O-4-nitrobenzoyl hydroxamates, esters,including methyl, ethyl and nitrophenyl esters, chloromethylketone andthe like.

[0064] Although endoproteases do not cleave terminal amino acids,endoproteases can cleave a carboxyl terminal blocking group attached viaa peptide bond to the carboxyl terminal amino acid of a peptidecontaining two or more amino acids (see Sarath et al., ibid.). If thecarboxyl terminal amino acid is the P1 amino acid of a scissile bond ina precursor protein, the carboxyl terminal blocking group mimics the P1′amino acid in that scissile bond. Moreover, endoprotease cleavage of thecarboxyl terminal blocking group mimics the cleavage of thecorresponding scissile bond in the precursor protein. Such carboxylterminal blocking groups include, for example, MCA, pNA, andpNA•Tosylate. An APP substrate which contains such a carboxyl terminalblocking group and an amino terminal blocking group can only be cleaved,if at all, by an endoprotease.

[0065] An APP substrate includes a secretase recognition site thatcontains a P1 amino acid of a scissile bond in an APP protein and acarboxyl terminal blocking group which replaces the P1′ amino acid inthat scissile bond. The APP substrate also contains one or more of theamino acids in the APP protein attached to the amino terminal side ofthe P1 amino acid. Such an APP substrate will bind a secretase whichbinds the corresponding scissile bond in the APP protein because thesubstrate contains the P1 amino acid, the primary determinant for thatbinding. For example, a β-secretase recognition site containing such acarboxyl terminal blocking group includes, for example, theVal-Lys-Met-MCA substrate in which the MCA group replaces the Asp P1′amino acid of the β-secretase scissile bond. Endoprotease cleavage ofthe Met-MCA peptide bond in that substrate is equivalent to endoproteasecleavage of the scissile bond Met-Asp of the β-secretase recognitionsite in the APP protein. Similarly a γ-secretase recognition site forthe Aβ₁₋₄₀ peptide includes, for example, the Gly-Val-Val-pNA substratein which the pNA group replaces the Ile P1′ amino acid of thecorresponding γ-secretase recognition site and endoprotease cleavage ofthe pNA group is equivalent to endoprotease cleavage of thecorresponding scissile bond in the APP protein. Similar substrates areenvisioned for the γ-secretase recognition site for the Aβ₁₋₄₂, andAβ₁₋₄₃ peptides and the α-secretase recognition site.

[0066] The APP substrate as discussed in the paragraph above can alsocontain an amino terminal blocking group. Only those secretases havingendoprotease activity will cleave that APP substrate and theendoprotease cleavage of the substrate will mimic that which occurs inthe APP protein. Examples of such APP substrates include, but are notlimited to, Z*Lys-Met-MCA, Z*Val-Lys-Met-MCA, Z*Val-Val-MCA,Z*Gly-Val-Val-MCA, Z*Ile-Ala-MCA, Z*Val-Ile-Ala-MCA, Z*Ala-Thr-MCA, andZ*Ile-Ala-Thr-MCA substrates. In these examples, Z represents the aminoterminal blocking group carbobenzoxy and the star (*) indicates anon-peptide bond between the Z and the adjacent amino acid. The MCArepresents the carboxyl terminal blocking groupaminomethylcourmarinamide and the dashes (-) represent peptide bondsbetween the MCA and the adjacent amino acid or between adjacent aminoacids.

[0067] Secretases having aminopeptidase activity can be assayed forusing an APP substrate that contains an amino acid of a secretaserecognition site and a carboxyl terminal blocking group. Examples ofsuch APP substrates include Met-MCA and Lys-MCA substrates from theβ-secretase recognition site. However, if such substrates contain onlyone amino acid, the substrate cannot be cleaved by an endoproteasebecause the only amino acid is an amino terminal amino acid. The Met-MCAand Lys-MCA substrates were used to identify β-secretase aminopeptidasesecretase activities (see Example IV).

[0068] An APP substrate often contains one or more labels thatfacilitate detection of the substrate or the APP derived product. Alabel can be an atom or a chemical moiety. Substrates containing a labelcan be made by methods known in the art. For example, radioactive atomssuch as ³H or ³²P can be attached to an APP substrate to detect an APPderived product. Also, heavy atoms or atom clusters such as, goldclusters can be attached. Moreover, fluorescent molecules such as,fluorescein, rhodamine, or green fluorescent protein, can be attached. Alabel can have more than one function. For example, the MCA is acarboxyl blocking group that is not fluorescent when bound in an APPsubstrate, is an APP derived product when cleaved by an endoproteasefrom a substrate, and is a label because, when MCA is cleaved from thesubstrate, it becomes fluorescent aminomethylcourmarinamide (AMC or freeMCA) which is detectable (Azaryan and Hook, Arch. Biochem. Biophys.314:171-177, (1994); and Azaryan et al., J. Biol. Chem. 270:8201-8208,which are incorporated herein by reference).

[0069] Cleavage of an APP substrate can be detected by the presence ofan APP derived product. The term “APP derived product” refers to aprotein, polypeptide, peptide or chemical moiety produced by proteolyticcleavage of an APP substrate. An APP derived product includes, forexample, an Aβ peptide, an α-APP fragment, a 10 kDa fragment, and AMC. Achemical moiety is the blocking group or label discussed above.

[0070] An APP derived product or an APP protein can be qualitatively orquantitatively detected using various methods. For example, theseproducts or proteins can be detected by an immunoassay using antibodiessuch as monoclonal or polyclonal antibodies against the Aβ₁₋₄₀. peptide,Aβ₁₋₄₂ peptide, Aβ₁₋₄₃ peptide, the amino terminal or the carboxylterminal regions of the APP proteins and the APP proteins. Suchantibodies are commercially available, for example, from PENINSULALABORATORIES, Belmont, Calif.; CALBIOCHEM, San Diego, Calif.; QCB,Hopkinton, Mass.; or IMMUNODYNAMICS, La Jolla, Calif.

[0071] SDS-PAGE electrophoresis and western blots can also be used todetect an APP derived product and an APP protein (see Example XII).Other methods include detecting a label on or from the APP derivedproduct or APP protein such as a radioactive or fluorescent label.Microsequencing, amino acid composition analysis, or mass spectrometryanalysis can also be used (see Example XV). Chromatography separationmethods based on physical parameters such as molecular weight, charge,or hydrophobicity can be used. Preferred chromatography methods includehigh pressure liquid chromatography (HPLC) and automated liquidchromatography (FPLC, PHARMACIA, Piscataway, N.J.). Spectrophotometricdetection methods such as UV absorbance at 280 nm or 210-215 nm, canalso be used. Known light or electron microscopic methods as well asfluorescent activated cell sorter methods also can be used to detect APPderived products and APP proteins. The quantitative fluorescenceanalysis using a fluorometer was used to detect the fluorescent AMCproduct produced by β-secretase cleavage of the Z*Val-Lys-Met-MCA,Met-MCA, and Lys-MCA (see Examples III, IV, VIII, and IX).

[0072]FIG. 2 shows the endoprotease cleavages that can occur in an APPsubstrate containing a β-secretase recognition site and amino andcarboxyl terminal blocking groups and how such cleavages can bedetected. In that figure, the three endoprotease cleavages of the APPsubstrate Z*Val-Lys-Met-MCA are shown (#1, #2, and #3). The Met-MCA bond(#3) mimics the scissile bond between the P1 and P1′ amino acids Met andAsp in the APP protein at the amino terminal end of the Aβ peptide.Endoprotease cleavage of the Met-MCA bond in the substrate is equivalentto endoprotease cleavage of the APP protein. That cleavage in the APPprotein would produce directly the amino terminal end of the Aβ peptide.That cleavage can be detected by the characteristic fluorescenceproduced by AMC (free MCA).

[0073] Endoprotease cleavage of the Lys-Met bond (#2) and the Val-Lysbond (#3) in the Z*Val-Lys-Met-MCA substrate produces a Met-MCA andLys-Met-MCA peptide, respectively. The corresponding endoproteasecleavages in the APP proteins would be distal to the amino terminal endof the Aβ peptide. However, such distal endoprotease cleavages can occurin vivo because, as discussed above, such cleavages followed byaminopeptidase cleavage of the flanking amino acids can produce theamino terminal end of the Aβ peptide.

[0074] The Met-MCA and Lys-Met-MCA peptides are not fluorescent, butcontain free amino terminal amino acids, which an aminopeptidase cancleave to liberate AMC. To insure that the endoprotease cleavages of theLys-Met and the Val-Lys bonds are detected, an aminopeptidase can beadded to an incubation solution to liberate AMC from the Met-MCA andLys-Met-MCA peptides. Known aminopeptidases include, for example,aminopeptidase M and methionine aminopeptidase (Mammalian Proteases, aGlossary and Bibliography, J. K. Mcdonald and A. J. Barrett, Ed.,Academic Press, Pub., p. 23-99, (1986)). In this manner, all theendoprotease cleavages of the Z*Val-Lys-Met-MCA substrate can bedetected.

[0075] Such methods were used to identify endoprotease activity of oneor more β-secretases in substantially purified vesicles (see ExamplesIII, VIII, and IX). In particular, a secretase in substantially purifiedvesicles was shown to cleave the Z*Val-Lys-Met-MCA substrate at a pH ofabout 4.0 to about 5.5 using these methods.

Methods of Isolating a Secretase

[0076] The present invention also is directed to a method of isolating asecretase using the assay described above to determine the proteolyticactivity of a secretase and isolating that secretase from substantiallypurified vesicles. Generally, the isolation is done by assaying theactivity of the secretase after each step in the isolation. Ifnecessary, the activity can be preserved during the isolation procedureusing methods such as those described above, including, for exampleisolating the secretase at a low temperature (e.g. 4° C.), or in thepresence of one or more of the above-described reducing or stabilizingagents.

[0077] The secretase is isolated based on its physical properties. Forexample, a secretase can be isolated based on its molecular weight andsize using gel filtration chromatography such as, Sephacryl S200,Sephadex G150, Superose 6 or 12, and Superdex 75 or 200 resinchromatography. A secretase can also be isolated based on its chargeusing ion-exchange chromatography such as DEAE-Sepharose, CM Sephadex,MonoQ, MonoS and MonoP resin chromatography. In addition, a secretasecan be isolated based on its water solubility using hydrophobicitychromatography such as phenyl Sepharose, butyl Sepharose and octylSepharose resin chromatography. Interactions between the secretase andhydroxyapatite can also be used for isolation using, for example,macro-prep hydroxyapatite, and Bio-Gel HT hydroxyapatite resins.

[0078] A secretase can also be isolated based on specific biochemicalproperties of the secretase using affinity chromatography. For example,the secretase can be isolated using APP substrate affinitychromatography under conditions in which the secretase binds the APPsubstrate but does not cleave it. Glycosylated secretases can beisolated using lectin affinity chromatography such as, concanavalinA-Sepharose, lentil lectin Sepharose, wheat germ lectin Sepharose resinchromatography. The proteolytic activity of sulfhydryl groups such asthose on cysteine amino acids can be used to isolate the secretasesusing thiol-propyl chromatography. Finally, the affinity of thesecretases for specific dyes can be used for separation such as,blue-Sepharose resin chromatography. Other affinity chromatographymethods include arginine-Sepharose, benzamidine Sepharose, glutathioneSepharose, lysine-Sepharose and chelating Sepharose resinchromatography. The secretases can also be isolated bynon-chromatographic fractionation methods using, for example, native gelelectrophoresis, analytical ultracentrifugation and differentialammonium sulfate precipitation methods (see Example XII).

[0079] Using such methods, alone or in combination, a secretase of theinvention can be isolated. The term “isolated” when used in reference toa secretase means that the secretase is relatively free of otherproteins, amino acids, lipids and other biological materials normallyassociated with a cell. Generally, an isolated secretase constitutes atleast about 50%, and usually about 70% to 80%, and often about 90 to 95%or more of the biological material in a sample. A secretase often isisolated such that it is free of other substances that affect thecleavage of an APP substrate, such as an inhibitor or activator protein.The extent to which the secretases are isolated using such methods canbe determined by known protein assays. For example, the amount ofprotein in the resulting chromatographic fractionation can bequantitated using the Lowry method and the specific activity can be usedto quantitate the isolation (see Example XIII). Alternatively, SDS-PAGEor two-dimensional gel electrophoresis and mass spectroscopy methods canbe used.

[0080] After initial isolation of a secretase, antibodies specific tothe secretase can be produced and secretases isolated usingimmunoaffinity chromatography. Such antibodies can be produced usingknown immunological methods including, for example, monoclonal antibodyand polyclonal antibody production methods (see Haylow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)).

[0081] The amino acid sequence of the secretase also can be determinedafter isolation of the secretase. For example, the amino acid sequenceof the secretase can be determined using peptide microsequencing methodsknown in the art (see “Current Protocals in Protein Science,” Vol. 1 and2, Coligan et al., Ed., (1997), John Wiley and Sons). Alternatively, thepartial amino acid sequence can be determined from fragments of thesecretase using mass spectrometry and Edman microsequencing methods(“Current Protocols in Protein Science,” Vol. 1 and 2, Coligan et al.,Ed., (1997), John Wiley and Sons). For example, the secretase can beisolated using an SDS-PAGE gel and stained with coomassie blue in thegel. The secretase in the gel can be subjected to in-gel trypticdigestion and the amount of protein determined by amino acid analysis.Tryptic peptide fragments can be separated by HPLC, and the amino acidsequence of each fragment determined by Edman microsequencing and massspectrometry methods. The amino acid sequence of the secretase can bedetermined from the amino acid sequences of the peptide fragments usingcomputer analysis of known amino acid sequences.

[0082] Based on the partial amino acid sequence of a secretase, the cDNAof the secretase can be cloned (see, for example, Molecular Cloning, aLaboratory Manual, Vol. 1, 2, and 3, Sambrook et al., Ed., Cold SpringHarbor Laboratory Press, Pub., (1989); and Current Protocols inMolecular Biology, Vol. 1, 2, and 3, Ausubel et al., Ed., WileyInterscience, Pub., (1997)). Briefly, partial, cloned secretase cDNAsare obtained by reverse transcription-polymerase chain reaction methods(RT-PCR) using oligonucleotides complementary to the partial amino acidsecretase sequences. The complementary oligonucleotides syntheticallysynthesized can contain either degenerate codons, including inosine, orbe optimized for mammalian cell use. The PCR-generated DNA fragment isanalyzed for nucleic acid sequences and restriction enzyme sequences,and overlapping sequences among the different PCR-generated DNAfragments are determined. Northern blot or RT-PCR analysis using thePCR-generated cDNAs, or complementary oligonucleotides, so produced areused to determine tissues that produce mRNAs encoding the secretase. AcDNA library from such tissues is constructed and screened using thePCR-generated secretase cDNA or the complementary oligonucleotides. Fromsuch screened cDNA libraries, the cDNA sequence encoding the full-lengthamino acid sequence of the secretase is determined.

[0083] The cDNA of a secretase can also be obtained by generatingantibodies against the partial amino acid sequences, screening cDNAexpression libraries with an anti-secretase antibody, and analyzing thenucleic acid sequences of such clones. The amino acid sequence of thesecretase can be deduced from the secretase cDNA sequence. Thefull-length cDNA can be cloned in an expression system such as in E.coli, Sf9 insect cells, yeast, or mammalian cell lines, and the activityof the expressed secretase determined to confirm that the cDNA encodes afunctional secretase.

[0084] Another method of obtaining the cDNA of a secretase is to clonethe secretase in a genetic screen for isolating the secretase cDNA usingthe bacteriophage 1 regulatory circuit, where the viral repressor isspecifically cleaved to initiate the lytic phase of bacteriophage toallow detection and isolation of plaques containing the secretasecDNA(s) (Sices and Kristie, Proc. Natl. Acad. Sci. USA 95:2828-2833,(1988)).

[0085] The gene(s) encoding a secretase can be isolated by screening agenomic library with the cDNAs encoding the partial or full lengthsecretase, or with the oligonucleotides that are complementary to asequence encoding a determined secretase amino acid sequence. Thenucleic acid sequence of the secretase genomic DNA is determined, andthe exon/intron structure of the secretase gene is determined bycomparing the DNA sequence of the gene to the nucleic acid sequence ofthe secretase cDNA.

[0086] Once the cDNA encoding a partial or full-length endogenoussecretase is obtained from one animal species, that cDNA can be used toobtain endogenous secretases from another animal species using knownmethods (Molecular Cloning, a Laboratory Manual, ibid.; and CurrentProtocols in Molecular Biology, ibid.). For example, the cDNA encodingthe partial bovine secretase can be used to obtain cDNAs encoding humansecretases. Briefly, a partial or full-length bovine cDNA, or a labeledcomplementary oligonucleotide, is used to isolate the human secretasecDNA by screening human cDNA libraries constructed from tissues thatcontain secretase mRNA, determined by northern blot or RT-PCR analyses.Alternatively, the human secretase cDNA can be obtained by searching theexpressed sequence tag database (EST) for human cDNA sequences similarto the bovine secretase cDNA. DNA sequencing of the resulting secretaseclones can be performed to determine the nucleic acid sequence encodingthe human secretase and the corresponding amino acid sequence can bededuced. The cDNA encoding the human secretase can be cloned in andexpressed by a suitable expression vector and the activity of theexpressed secretase can be determined. The genes encoding the humansecretase can be cloned as described herein.

[0087] The nucleic acid sequence of a secretase can also be used toproduce the secretase using known recombinant methods (MolecularCloning, a Laboratory Manual, ibid.; and Current Protocols in MolecularBiology, ibid.). The cDNA encoding the secretase can be inserted into anappropriate expression vector and the expression vector introduced intoan appropriate host as described herein. Expression of the secretase bythe host is stimulated by expression of a vector promotor.

Methods of Screening for Agents that Affect the Proteolytic Activity ofa Secretase

[0088] Another aspect of the invention is a method of selecting an agentthat alters the cleavage of an APP substrate by a secretase. Suchagents, particularly those that decrease the cleavage by the β-secretaseand γ-secretases or that increase the cleavage by the α-secretase, areuseful for developing drugs that prevent or treat AD. Agents havingdivergent chemical structures can be assayed using such methodsincluding, for example, small organic molecules that optionally containheteroatoms or metals, amino acids, peptides, polypeptides, proteins,peptidomimetics, nucleic acids, carbohydrates, glycoproteins, lipids,and lipoproteins.

[0089] The method is based on comparing the APP substrate cleavage, orthe APP protein, or APP derived product production that occurs with andwithout an agent. This is achieved by determining the APP substratecleavage or the APP protein or the APP derived product produced in afirst incubation or culture solution lacking the agent and comparingthat result with that which occurs in a second incubation or culturesolution containing the agent. The first and second incubation orculture solutions can be different solutions or the same solution towhich the agent is added or removed. The APP substrate cleavage, the APPprotein, and the APP derived product can be assayed using the methodsdescribed herein. The concentration of the agent can vary due toparameters known in the art such as the hydrophobicity, charge, size andpotency of the agent, but typically is about a 10⁻⁹ to 10⁻³ M.

[0090] Agents are selected that alter the cleavage of an APP substrateor production of an APP protein or an APP derived product. The cleavageor production is altered if the agent causes a significant change in thecleavage or production relative to that which occurs without the agent.A significant change can be determined using a variety of qualitative orquantitative methods, such as, for example, by a visual or statisticalanalysis of the comparison data. For example, the mean amounts of an APPderived product obtained with and without the agent can be analyzedusing a two-sided Student's t-test and a p≧0.02 or greater, andpreferably a p≧0.05, in that test can be indicative of a significantdifference.

[0091] Often agents are screened using substantially pure vesicles asthe source of the secretase. But substantially pure vesicles cancontain, in addition to secretases, other substances that affect thecleavage of an APP substrate, such as the presenilin 1 protein. Thus, ascreen using such vesicles selects for agents that directly orindirectly alter the cleavage. An agent can directly affect the cleavageby, for example, inhibiting the binding of an APP substrate to asecretase. But an agent can also indirectly alter the cleavage byaffecting an inhibitor or activator substance which in turn affects theactivity of the secretase. For example, proteases may be present in thevesicle that produce the secretase from a precursor protein or thatdegrade the secretase. An agent thus can indirectly affect the secretaseactivity by affecting the proteases which produce or degrade thesecretase. Often permeablized chromaffin vesicles and an APP protein, Aβpeptide, Z*Val-Lys-Met-MCA, Z*Gly-Val-Val-MCA, Z*Val-Ile-Ala-MCA, orZ*Ile-Ala-Thr-MCA substrate are used in the assay.

[0092] An isolated secretase, obtained as described above, can also beused to select for agents that affect the activity of the secretase.Using an isolated secretase free of other substances that affect thecleavage of an APP substrate, agents can be selected that directlyaffect cleavage of the APP substrate. The affect of an agent on such anisolated secretase and on substantially purified vesicles can becompared to determine the direct and indirect affects of the agent.Moreover, that comparison can be used to determine if the vesiclescontain inhibitors or activators of the secretase removed duringisolation of the secretase.

[0093] The protease class to which an isolated secretase belongs can bedetermined using agents known to selectively inhibit different classesof proteases. For example, E-64c, cystatin, and p-mercuribenzoateinhibit cysteine proteases; phenylmethylsulfonyl fluoride (PMSF),soybean trypsin inhibitor, and α₁-antitrypsin inhibit serine proteases;ethylenediaminetetraacetic acid (EDTA) and 1,10-O-phenanthroline inhibitmetalloproteases; and pepstatin A inhibits aspartyl proteases. (SeeExamples XI and XIV).

[0094] In another method, a cell containing vesicles having theproteolytic activity of a secretase is used to select for an agent.Cells containing such vesicles can be identified using the methodsdescribed above to determine the proteolytic activity of a secretase inthe vesicles. The cells are cultured in a first culture solution withoutthe agent and in a second culture solution with the agent and theproduction of an APP protein or an APP derived product by the cell,especially an Aβ peptide, α-APP fragment or 10 kDa fragment, in thefirst and second culture solution compared.

[0095] A problem with using transformed cell cultures or cell lines toselect agents is that the agents may be ineffective in vivo becausecells in culture can process a protein in a manner unrelated to thatwhich occurs in vivo. Thus, agents that affect the processing of suchcells are ineffective because the processing that they affect does notoccur in vivo. The cell based method provided in the present inventionavoids this problem by selecting cells determined to contain vesiclesthat have the proteolytic activity of a secretase. As such, the methodinsures that the cells process the APP protein in the cell organelle inwhich that processing occurs in vivo

[0096] A cell used in this method can be obtained from a varietysources. For example, disassociated cells maintained in a primaryculture can be used in the method. Such disassociated cells can bemaintained in a primary culture using known methods (see, for example,Hook et al., ibid.; and Tezapsidis et al., ibid.). Disassociated cellshave the advantage of retaining many of the functional characteristicsthat they have in the tissue that they are obtained from. But primarycultures of disassociated cells generally die after a period of time.Cell lines, transformed cells and cloned cells, on the other hand, havethe advantage of being immortal. But such cells are known to oftenabnormally process proteins. As such, it is particularly important touse immortalized cells that are determined to contain vesicles in whichthe proteolytic activity of a secretase occurs so as to insure that thecells are processing the APP protein in the same manner as in vivo.Various cell transformation methods can be used to obtain such cells(see for example, Alarid et al. Development, 122(10):3319-29, (1996);and Schecter et al., Neuroendocrinology, 56(3):300-11, (1992), which areincorporated herein by reference). A chromaffin cell, either obtained bydisassociation or by transformation, is often used in this method.

[0097] In the cell based assay of the present invention, the agent isoften present when the cells are producing an APP derived productbecause some agents are known to only affect a protease in a cell whenthe protease is producing a product. For example, agents are known toinhibit enkephalin production in chromaffin cells only when thechromaffin cells are actively producing enkephalin (Tezapsidis et al.,ibid.). Various methods can induce cells to produce proteolyticallyprocessed peptides in vesicles. For example, proteolytic processing canbe induced by exocytosis. Exocytosis can be induced by various meansincluding, for example, by increasing the extracellular potassiumchloride concentration or by binding nicotinic cholinergic receptors oncells with nicotine. Proteolytic processing of the Aβ peptides can alsobe induced by stimulating protein kinase with phorbol esters (Koo,Molec. Medicine, 3:204-211, (1997); and LeBanc et al., J. Neurosci.,18:2908-2913, (1998)).

[0098] For example, as shown in Example VII, chromaffin cells can beinduced to produce an Aβ peptide by culturing the cells in potassiumchloride (about 5 to 500 mM), nicotine (about 10⁻³ to 10⁻⁶ M), orphorbol ester (about 10⁻³ to 10⁻⁶ M) for a sufficient amount of time tostimulate production (about 1 to 72 hours for the nicotine and potassiumchloride and about 12 to 96 hours for the phorbol ester). During activeproduction of the Aβ peptide by the cells, an agent is incubated withthe chromaffin cells under appropriate conditions and for an appropriateamount of time (e.g. about 2 to 8 hours). The cells can then be lysedand the production of an Aβ peptide with and without the agent compared.To facilitate that comparison, a protease inhibitor such as,chymostatin, leupeptin, and soybean trypsin inhibitor (STI), can beadded when cells are lysed to prevent non-specific digestion of the Aβpeptide by non-specific proteases released by cell lysis.

[0099] The cell based assay can be used to select an agent that affectscell expression. For example, the expression of a nucleic acid thatencodes a secretase can be tested in such an assay. Inhibitors of genetranscription, such as actinomycin D or an antisense nucleic acid, oragents that modify protein transcription factors that regulate geneexpression, such as steroids, also can be tested. The cell based assaycan also be used to select agents that affect protein processing,including those affecting RNA splicing, RNA polyadenylation, RNAediting, protein translation, signal peptidase processing, proteinfolding including chaperone-mediated folding, disulfide bond formation,glycosylation, phosphorylation, covalent modification includingmethylation, prenylation, and acylation, and association with endogenousprotein factors that modify secretase activity.

[0100] Agents found to alter cleavage of an APP substrate can beevaluated in vivo using transgenic AD animal models. Transgenic animalmodels have been developed in which the animals have brain amyloidplaques containing Aβ peptides and, in some models, exhibit cognitivedeficits such as excessive memory loss. Exemplary transgenic animalsinclude mice that contain the Indiana mutation of the human APP cDNAunder the control of the PDGF promoter (Johnson-Wood et al., Proc. Natl.Acad. Sci., USA, 94:1550-1555, (1997)). These mice express increasedlevels of brain Aβ peptides and amyloid plaques and show cognitivedeficits. Another exemplary transgenic animal is a mouse straincontaining the Swedish mutation of the human APP-695 cDNA with thehamster PrP promoter (Hsiao, J. Neural Transmission, 49:135-144,(1997)). These mice express increased levels of brain Aβ peptides, haveamyloid plaques and are memory impaired.

[0101] Agents can be administered to such animals using methods known inthe art, particularly those methods that result in the agent traversingthe blood brain barrier. For example, such agents can be administered bydirect injection into the central nervous system or by administrationwith a minipump. Agents that naturally traverse the blood brain barriercan be systematically administered by intravenous, subcutaneous, or oralroutes. Such agents can be administered in effective doses which forexample can range from 0.001 to 10 mg/kg body weight. Agents can beadministered prophylactically or therapeutically in single or multipledose schedules.

[0102] Agents can be assayed by histopathological examination of thebrains from such transgenic animals. For example, quantitative,microscopic analysis of amyloid plaque formation can be used todetermine the effect of the agent. Agents which reduce the size orfrequency of amyloid plaques are preferred. In addition, agents can beassayed by measuring brain levels of Aβ₁₋₄₀, Aβ₁₋₄₂, or Aβ₁₋₄₃ byradioimmunoassay or ELISA. Agents that reduce Aβ₁₋₄₀, Aβ₁₋₄₂, or Aβ₁₋₄₃levels are preferred. Agents also can be assayed for their effect on thecognitive behavior of such animals using known methods. For example, thememory capability of mice can be determined using the water maize test.Agents which enhance the memory capability are preferred.

[0103] Agents that effectively reduce or inhibit Aβ peptide productionor amyloid plaque formation or increase memory in any of the methodsdescribed above can be used to treat or prevent AD. Persons identifiedas probable AD patients by known medical methods can be administeredsuch agents. Also, people diagnosed as having a high probability ofdeveloping AD can be administered such agents. Patients are assessed forimprovement in cognitive abilities. Upon autopsy, brain tissue isassessed for amyloid plaques and Aβ levels. Agents are administered byknown methods such as those described above for the animal model.

[0104] Agents that effectively reduce or inhibit β B peptide productionor amyloid plaque formation or increase memory can also be used toenhance memory function of people, especially the elderly. People can beadministered such agents and assayed for improved memory capability.Agents can be administered by known methods such as those describedabove for the in vivo assay.

[0105] The following examples are intended to illustrate but not limitthe present invention.

EXAMPLE I Isolation of Chromaffin Vesicles

[0106] Chromaffin vesicles were isolated from fresh bovine adrenalmedulla by discontinuous sucrose gradient centrifugation (Krieger etal., Biochemistry, 31, 4223-4231, (1992); Yasothornsrikul et al., J.Neurochem. 70, 153-163, (1998)). Briefly, fresh bovine adrenal glandswere dissected to obtain the medulla region. These medulla from 40glands were homogenized in 200-250 ml ice-cold 0.32 M sucrose, and thehomogenate was centrifuged at 1,500 rpm in a GSA rotor (Sorvallcentrifuge) for 20 minutes at 4° C.

[0107] The resultant supernatant was collected and centrifuged at 8,800rpm in a GSA rotor (Sorvall centrifuge) for 20 minutes at 4° C. toobtain a pellet of chromaffin vesicles. The pellet of chromaffinvesicles was washed three times in 0.32 M sucrose. Each wash consistedof resuspending the pellet of chromaffin vesicles with an equal volume(same volume as original homogenate) of 0.32 M sucrose andcentrifugation at 8,800 rpm in a GSA rotor to collect the vesicles asthe pellet.

[0108] After washing, the chromaffin vesicles were resuspended in 120 mlof 0.32 M sucrose and subjected to discontinuous sucrose gradientcentrifugation. For that centrifugation, 10 ml of the washed chromaffinvesicle suspension was layered on top of 25 ml of 1.6 M sucrose in eachof 12 centrifuge tubes. The 12 tubes of sucrose gradient werecentrifuged in a SW28 rotor at 25,000 rpm for 120 minutes at 40° C. Thepellets of isolated chromaffin vesicles from 12 tubes were resuspendedin 12 ml of 0.015 M KCl with a glass-glass homogenizer, and stored at−700° C., prior to use. A chromaffin vesicle lysate was prepared byfreeze-thawing the isolated chromaffin vesicles in the 15 mM KCl.

EXAMPLE II Assay for Chromaffin Vesicles

[0109] The chromaffin vesicles in the Example I preparation were assayedfor the chromaffin vesicle markers (Met)enkephalin, catecholamines, thelysosomal marker acid phosphatase and total protein. Fractionscontaining the highest amount of chromaffin vesicle markers wereidentified as chromaffin vesicles. The homogeneity of the chromaffinvesicles was approximately 99% as assayed by the proteolytic activity ofthe chromaffin vesicle markers (Met)enkephalin and catecholamines andthe absence of the lysosomal marker acid phosphatase. Electronmicroscopy showed that uniform, homogeneous, and intact chromaffinvesicles were isolated. The chromaffin vesicles were purifiedapproximately 8-fold from the cell homogenate based on the measurementof the picograms of (Met)enkephalin per microgram of protein in thesamples.

EXAMPLE III β-secretase Endoprotease Activity

[0110] The APP substrate, Z*Val-Lys-Met-MCA, was used to identify aβ-secretase based on endoprotease activity. That substrate wascommercially obtained and had a purity of 99% or better as determined bythe manufacturers (PENINSULA LABORATORIES, Belmont, Calif. and PHOENIXLABORATORIES, Mountain View, Calif.;).

[0111] The β-secretase endoprotease activity was identified byincubating the chromaffin vesicle lysate (2-10 μl of 10-20 mgprotein/ml) with the Z*Val-Lys-Met-MCA substrate (100 μM finalconcentration) and detecting AMC fluorescence. The chromaffin vesiclelysate was prepared as described in Example I. The endoprotease activitywas determined as a function of pH by varying the pH of the incubationsolution between 3.0 to 8.0 in 0.5 pH increments. Citric acid, sodiumphosphate, and Tris-HCl buffers (100 mM final concentration) were usedto adjust the pH of the incubation solutions between 3.0 to 5.5, 6.0 to7.5, and 8.0, respectively. Duplicate samples at each pH increment (100μl each) were distributed among 22 wells in a covered microtiter wellplate and incubated at 37° C. for 8 hours in a water bath.

[0112] As discussed above, endoprotease cleavage between the Met-MCAbond in the Z*Val-Lys-Met-MCA substrate produces fluorescent AMC, butendoprotease cleavage between the Lys-Met or Val-Lys bonds in thatsubstrate produces non-fluorescent Lys-Met-MCA and Met-MCA peptides. Toinsure that the latter two endoprotease cleavages were detected,aminopeptidase M (20 μg/ml final concentration, BOEHRINGER MANNHEIM) wasadded to each incubation solutions to produce fluorescent AMC from theLys-Met-MCA and Met-MCA peptides. Prior to adding the aminopeptidase M,each incubation solution was adjusted to a pH 8.3 using Tris-HCl becauseaminopeptidase M functions at a basic pH. A second incubation at 37° C.for 1 hour in the water bath was conducted to complete theaminopeptidase M reaction.

[0113] Upon termination of that second incubation, AMC fluorescence wasassayed using a fluorometer (IDEXX fluorometer, FCA FluorescenceConcentration Analyzer, cat. no. 10-105-2, BAXTER HEALTH CARE CORP.,Mundelein, Ill.) at excitation and emission wavelengths of 365 and 450nm, respectively. Standard AMC concentrations were also measured toquantitate relative fluorescence with the molar amount (pmol) of AMCgenerated by the secretase. The resulting AMC fluorescence reflects theendoprotease activity in cleaving either the Met-MCA, Lys-Met. andVal-Lys bonds in the Z*Val-Lys-Met-MCA substrate.

[0114] The AMC fluorescence was plotted as a function of pH and is shownin FIG. 3. Analysis of that plot shows a principal β-secretaseendoprotease activity having a pH optimum of about 4.5-5.0. In addition,the plot shows two lesser β-secretase endoprotease activities having pHoptimums of about pH 3.5 and 6.0-6.5.

EXAMPLE IV β-secretase Aminopeptidase Activity

[0115] The APP substrates, Met-MCA, and Lys-MCA, were used to identify aβ-secretase based on aminopeptidase activity. Those substrates werecommercially obtained and had a purity of 99% or greater as determinedby the manufacturers (PENINSULA LABORATORIES, Belmont, Calif. andPHOENIX LABORATORIES, Mountain View, Calif.).

[0116] The β-secretase Met aminopeptidase activity was identified byincubating the chromaffin vesicle lysate (5 μl of 10-15 mg/ml) with theMet-MCA substrate (100 μM final concentration) and detecting theresulting AMC fluorescence. The chromaffin vesicle lysate was preparedas described in Example I. The aminopeptidase activity was determined asfunction of pH by varying the pH of the incubation solution between 3.0to 8.0 in 0.5 pH increments. Citric acid, sodium phosphate, and Tris-HClbuffers (100 mM final concentration) were used to adjust the pH of theincubation solutions between 3.0 to 5.5, 6.0 to 7.5, and 8.0,respectively. Duplicate samples at each pH increment (100 μl each) weredistributed among 22 wells in a covered microtiter well plate andincubated at 37° C. for 4 hours in a humidified incubator.

[0117] Similarly, the β-secretase Lys aminopeptidase activity wasidentified by incubating the chromaffin vesicle lysate (5 μl of 10-15mg/ml) with the Lys-MCA substrate (100 μM final concentration) anddetecting the resulting AMC fluorescence. The incubation was identicalto that described for the Met aminopeptidase assay except that theincubation time was 2 hours long.

[0118] Upon termination of the incubations, AMC fluorescence was assayedas described above. The resulting AMC fluorescence indicatingβ-secretase Met and Lys aminopeptidase activities was plotted as afunction of pH and is shown in FIGS. 4 and 5, respectively.

[0119] Analysis of FIG. 4 shows a β-secretase Met aminopeptidaseactivity having a pH optimum of about 5.5-6.5. Similarly, analysis ofFIG. 5 shows a β-secretase Lys aminopeptidase activity having a pHoptimum of about 6.0-7.0.

EXAMPLE V Identification of Aβ peptides

[0120] The chromaffin vesicle lysate was analyzed for the proteolyticactivity of Aβ peptides using commercially available polyclonal andmonoclonal antibodies against the Aβ₁₋₄₀ and Aβ₁₋₄₂ (PENINSULALABORATORIES, Belmont, Calif.; and QCB, Hopinton, Mass., respectively)in known radioimmunoassay (RIA) and ELISA methods. The chromaffinvesicles contained Aβ₁₋₄₀ at 0.051 pg/ug protein as determined by RIAand a detectable amount of Aβ₁₋₄₂ as determined by ELISA.

EXAMPLE VI APP Protein Distribution in Chromaffin Cells

[0121] The distribution of APP protein in chromaffin cells wasdetermined using a monoclonal antibody directed against the aminoterminal region of the APP protein (Anti-Alzheimer precursor protein A4,clone #22C11, BOEHRINGER MANNHEIM, Indianapolis, Ind.) in establishedimmunofluorescent cytological methods. Fluorescent light microscopicanalysis of chromaffin cells stained by this method showed that the APPprotein was localized in the chromaffin vesicles and not in the cellnucleus.

EXAMPLE VII Aβ-peptide Secretion by Chromaffin Cells

[0122] Primary chromaffin cell cultures containing approximately 2million cells in each culture were produced using established methods(Hook et al., ibid.; and Tezapsidis et al., ibid.). Exocytosis of thecontents of the vesicles in such cells was induced by exposing the cellsto KCl (50 mM) or nicotine (10 μM) for 15 minutes. The media was removedfrom the cells and the Aβ₁₋₄₀ peptide in the media was determined usingthe RIA assay described in Example V. The KCl and nicotine exposurecaused an approximately 350-fold and 550-fold increase in theconcentration of Aβ₁₋₄₀ peptide in the media, respectively, relative tothat of a control media from a culture identically processed but whichdid not receive KCL or nicotine. The results show that chromaffin cellsexocytosis results in the secretion of Aβ peptide.

EXAMPLE VIII Effect of Reducing Agents on β-secretase EndoproteaseActivity in Chromaffin Vesicles

[0123] The effect of the reducing agent dithiothreitol (DTT) onβ-secretase endoprotease activity was determined using the assaydescribed in Example III. Briefly, the lysed vesicles were incubatedwith the substrate Z*Val-Lys-Met-MCA in the presence or absence of 1 mMDTT and the resulting fluorescence plotted as a function of pH. Bothwith and without DTT, β-secretase endoprotease activity was detected andin both cases that activity had pH optimum of about 4.0 to 6.0, which isconsistent with the intravesicular pH of chromaffin vesicles. But theDTT resulted in a significant increase in the β-secretase endoproteaseactivity, approximately 5-fold (see Figure 6). These results show thatDTT, although not essential, significantly increases β-secretaseendoprotease activity.

EXAMPLE IX Effect of Aminopeptidase M on β-secretase EndoproteaseActivity in Chromaffin Vesicles

[0124] The effect of the aminopeptidase M and the basic pH buffer usedin the β-secretase endoprotease activity assay was determined. The assaywas conducted as described in Example VIII with DTT. Three assays wereconducted, one with aminopeptidase M in its basic pH buffer, anotherwith the basic pH buffer but not aminopeptidase M, and a third withouteither the buffer or the aminopeptidase M. Briefly, the chromaffinvesicle lysate and the substrate Z*Val-Lys-Met-MCA were incubated for 30minutes at a specified pH and the resulting fluorescence measured. Theaminopeptidase M in the basic pH buffer or that buffer alone (finalconcentration of 75 mM Tris-HCl pH 8.2) was added to the assay andincubated an additional 60 minutes at 37° C. The resulting fluorescencewas plotted as a function of pH, which showed that β-secretaseendoprotease activity occurred in the 3 assays (see FIG. 7). The assayconducted with aminopeptidase M and its basic pH buffer and that of thecontrol assay having just the basic pH buffer produced approximately thesame amount of fluorescence. This result is consistent with thatobtained in Example IV, which showed that chromaffin vesicles contain anendogenous β-secretase methionine and lysine aminopeptidase.

EXAMPLE X β-secretase Endoprotease Activity Obtained During Isolation ofChromaffin Vesicles

[0125] The β-secretase endoprotease activity of fractions obtainedduring the isolation procedure described in Example I was determined atthe pH optimum of 5.5, with and without DTT using the assay described inExample VII. The ratio of those activities (with/without DTT) wascalculated and the ratios obtained for the fraction shown in Table I.TABLE I FRACTION RATIO Adrenal Medulla Homgenate 4.7 Pellet from 1,500rpm Centrifugation (nuclear 11.6 fraction) Pellet from 1st 8,800 rpmCentrifugation (crude 3.2 vesicle fraction) Pellet from 2nd 8,800 rpmCentrifugation (washed 6.3 vesicle fraction) Pellet from 25,000 rpmDiscontinuous Gradient 11.0 Centrifugation (vesicle fraction)

[0126] The results show that β-secretase endoprotease activity isenriched in the nuclear fraction and the vesicle fraction. But, asdescribed in Example VI, only the chromaffin vesicles contain the APPprotein, and thus only in that fraction does the protease havingβ-secretase endoprotease activity also have access to the APP proteinsubstrate.

EXAMPLE XI Protease Inhibitors of β-secretase Endoprotease Activity inChromaffin Vesicle Lysate

[0127] The effect of various protease inhibitors on β-secretaseendoprotease activity in the lysate was determined at the pH optimum 5.5in the assay described in Example IX containing aminopeptidase M.Protease inhibitors specific for various protease classes were used. Theprotease inhibitor was added to each assay at the start of the reactionat the appropriate concentration. The extent of inhibition was expressedas a percentage of the activity without the inhibitor (control).Triplicate assays varied by less than 10%. The results are shown inTable II. TABLE II INHIBITOR PROTEASE CLASS (Concentration) % CONTROLControl None 100 Cysteine E64c (10 μM) 0 Cysteine pHMB (1 mM) 35 SerinePMSF (100 μM) 58 Serine Chymostatin (10 μM) 11 Aspartyl Pepstatin A (10μM) 78 Metallo EDTA (1 mM) 100 Metallo EGTA (1 mM) 99 NonspecificLeupeptin (100 μM) 0

[0128] The results show that the β-secretase endoprotease activity inthe chromaffin vesicle lysate was completely inhibited by the cysteineprotease class inhibitor E64c, and the nonspecific protease inhibitorleupeptin. The serine protease class inhibitor chymostatin and thecysteine protease inhibitor pHMB greatly inhibited activity. The apartylprotease class inhibitor pepstatin A slightly inhibited the activity andthe metallo protease class inhibitors did not inhibit activity.

EXAMPLE XII Isolation of β-Secretases from Chromaffin Vesicles

[0129] The chromaffin vesicle lysate was separated into 2 β-secretaseendoprotease activity peaks (referred to as “Peak I” and “Peak II”).Peak I had about 3 times the total activity of Peak II and a differentβ-secretase endoprotease activity than did Peak II. The Peak I activitywas very sensitive to the presence of aminopeptidase M in the assaywhereas the Peak II activity was relatively insensitive toaminopeptidase M.

[0130] The Peak I center and range of activities had molecular weightsof about 185 kDa, and about 180 to 200 kDa, respectively. Peak I wasfound to be a protease complex having a broad band of activity asdetermined by a native PAGE activity assay and 3 distinct activitiescorresponding to molecular weights of about 88, 81, and 61 kDa, in anon-reducing SDS-PAGE activity assay. Peak I was found to contain 3proteins having molecular weights of about 88, 81, and 36 kDa, and 4proteins having molecular weights of about 66, 60, 33, and 29 kDa, in anon-reducing and a reducing SDS-PAGE stained for proteins, respectively.

[0131] Peak II had a center and range of activities having molecularweights of about 65 kDa, and about 50 to 90 kDa, respectively. Peak IIcontained 2 proteins having different net electronegative charges andβ-secretase endoprotease activity (referred to as “Peak II-A” and “PeakII-B”).

Isolation of Peaks I and II and Characterization of the β-SECRETASEEndoprotease Activites in those Peaks

[0132] The procedure used to isolate Peaks I and II is diagramed in FIG.8. The β-secretase endoprotease activity with and without aminopeptidaseM was determined after each isolation step using the assay described inExample IX. Isolation steps that enriched that activity were selected.The total and specific activities after each isolation step aresummarized in Example XIII. The β-secretase aminopeptidase activity wasdetermined by the assay described in Example IV.

[0133] Preliminary experiments indicated that the β-secretase is presentin chromaffin vesicles at a relatively low concentration. Thus, a verylarge number of bovine adrenal glands, approximately 2400, was used sothat a sufficient amount the β-secretase could be obtained for analysis.Using the methods described in Example I, numerous chromaffin vesiclelysate preparations were made over a period of approximately 6 monthsand pooled.

[0134] A soluble extract and membrane pellet from the pooled lysate wasmade by ultracentrifugation at approximately 100,000×g. The bulk of theactivity was in the soluble extract and was aminopeptidase insensitive(see Krieger, T. K. and Hook, V. Y. H. J. Biol. Chem. 266, 8376-8383,(1991). As such, it was concluded that the β-secretase endoproteaseactivity was not bound to the chromaffin vesicle membranes.

[0135] The soluble extract was separated by concanavalin A-Sepharoseresin chromatography (referred to as “Con A”) into bound and unboundfractions. The Con-A bound fraction was subsequently eluted usingalpha-methylmannoside (referred to as the “eluted Con-A bound fraction”)and contained the bulk of the β-secretase endoprotease activity, but noβ-secretase aminopeptidase activity. The unbound fraction (referred toas the “Con-A unbound fraction”), in contrast, contained β-secretasemethionine and lysine aminopeptidase activity, but little β-secretaseendoprotease activity. The Con-A step thus separated the endogenousβ-secretase endoprotease and aminopeptidase activities (see Krieger, T.K. and Hook, V. Y. H., ibid.).

[0136] The contents of the eluted Con-A bound fraction were fractionedaccording to molecular size using a Sephacryl S200 column (Krieger, T.K. and Hook, V. Y. H. ibid.) . That resulted in the Peak I and Peak IIβ-secretase endoprotease activities. The Peak I center and range ofactivities corresponded to proteins having molecular weights ofapproximately 185 kDa, and 180 to 200 kDa, respectively. The Peak IIcenter and range of activities corresponded to proteins having molecularweights of approximately 65, and 50 to 90 kDa, respectively (see FIG.9).

[0137] Peak I had more than 3 times the total activity of Peak II, butthe Peak I activity without aminopeptidase M was only about 5% of thatproduced with the aminopeptidase. Thus, Peak I was aminopeptidasesensitive. Since Peak I alone did not produce much fluorescence, themajority of the Peak I activity does not cleave the Met-MCA bond in theZ*Val-Lys-Met-MCA substrate because cleavage of that bond must occur toproduce fluorescent free MCA. But since the addition of aminopeptidase Mproduced a significant amount of fluorescence, the majority of the PeakI activity must endoproteolytically cleave that substrate because thatcleavage must occur, for reasons discussed above, in order for theaminopeptidase M to cleave the Met-MCA bond and the Lys-Met bond andproduce fluorescent free MCA. The Peak I activity thus must cleave theLys-Met or the Val-Lys bond because those are the only other peptidebonds in the substrate that can be cleaved. Moreover, the fact thataminopeptidase M must be added to Peak I to detect activity confirmsthat the Con-A isolation step removed most of the endogenousaminopeptidases from the eluted Con-A bound fraction.

[0138] As discussed above, the Met-MCA bond in the Z*Val-Lys-Met-MCAsubstrate is a mimic of the β-secretase scissile bond Met-Asp in the APPprotein. As such, failure of the Peak I β-secretase endoprotease tocleave the Met-MCA bond means that it also does not cleave theβ-secretase scissile bond. Rather, as discussed below, the majority ofthe Peak I β-secretase endoprotease activity preferentially cleaves theLys-Met in the β-secretase recognition site. Thus, for the Peak Iβ-secretase endoprotease to produce the amino terminal end of the Aβpeptide from an APP protein, several cleavages must occur. For example,the Peak I β-secretase endoprotease can cleave the Lys-Met bond adjacentto the β-secretase scissile bond and, second, an endogenous β-secretaseaminopeptidase can cleave off the amino terminal Met in the β-secretasescissile bond Met-Asp to produce the amino terminal end of the Aβpeptide. Alternatively, the Peak I β-secretase endoprotease can cleavethe Val-Lys bond and an endogenous β-secretase aminopeptidase(s)subsequently cleave off the Lys and Met amino acids and produce theamino terminal end of the Aβ peptide.

[0139] In contrast, Peak II was relatively aminopeptidase insensitive asits activity without aminopeptidase M was about 84% of that with theaminopeptidase. Thus, the majority of the Peak II activity cleaves theMet-MCA bond in the substrate Z*Val-Lys-Met-MCA directly because Peak IIalone produces fluorescent free MCA. As the Met-MCA bond is a mimic ofthe β-secretase scissile bond, the majority of Peak II β-secretaseendoprotease activity also cleaves the β-secretase scissile bond whichcan directly produce the amino terminal end of the Aβ peptide.

[0140] But the modest increase in the fluorescence produced by Peak IIwith aminopeptidase M indicates that some of the Peak II activity alsocleaves the Lys-Met or the Val-Lys bond in the Z*Val-Lys-Met-MCAsubstrate for reasons described above regarding Peak I. Similarly, someof the Peak II activity also can produce the amino terminal end of theAβ peptide by a combination of endoprotease and aminopeptidase cleavagesas discussed above regarding Peak I.

[0141] These results demonstrate that multiple β-secretases are involvedin producing an Aβ peptide from an APP protein.

Isolation of β-SECRETASES from Peak I

[0142] The procedure used to isolate the β-secretases from Peak I isdiagramed in FIG. 10. The Sephacryl S200 column fractions containing thePeak I β-secretase endoprotease activity were pooled (referred to as the“Peak I Sephacryl S200 fraction”) and chromatographed on achromatofocusing Polybuffer Exchange 94 column (PHARMACIA, Piscataway,N.J., referred to here as “CF”). The CF fractions containing theβ-secretase endoprotease activity were pooled and concentrated withbuffer exchange to 100 mM citric acid-NaOH, pH 4.5, using an AMICONultrafiltration apparatus equipped with a YM 10 membrane. (referred toas the “Peak I CF fraction” or “CF fraction,” see Krieger, T. K. andHook, V. Y. H., ibid.).

[0143] The Peak I CF fraction, in turn, was purified using cation Mono Sexchange chromatography by FPLC (referred to as “Mono S”). The CFfraction was loaded onto a Mono S ion exchange FPLC column (1 ml HiTrapcolumn SP, PHARMACIA, Piscataway, N.J.) that was equilibrated with 100mM citric acid-NaOH, pH 4.5 (referred to as “buffer A”). The column waseluted with a NaCl gradient generated with a buffer consisting of 100 mMcitric acid-NaOH, pH 4.5, 2.0 M NaCl (referred to as “buffer B”), withthe gradient consisting of 0% buffer B at 1-15 min., 0-25% buffer B at15-45 min., 25-100% buffer B at 45-50 min., 100% buffer B at 50-55 min.,100-0% buffer B at 55-60 min., and 0% buffer B at 60-75 min., with aflow rate of 1 ml/min. Fractions containing β-secretase endoproteaseactivity were pooled and concentrated by AMICON ultrafiltration withbuffer exchange to 100 mM citric acid-NaOH, pH 4.5 (referred to as the“Peak I Mono S fraction” or “Mono S fraction”).

[0144] The Mono S fraction was further analyzed by variouspolyacrylamide gel electrophoresis (PAGE) methods. Referring in FIG. 10,one such method was a “native PAGE in gel activity assay,” whichdetermined the β-secretase endoprotease activity of the Mono S fractionin the PAGE gel. In this assay, the proteins are first separated byelectrophoresis and then allowed to proteolytically react with asuitable substrate in the gel. Proteins having proteolytic activity areidentified by the formation of a cleavage product in the gel. A suitablesubstrate and cleavage product for detecting a secretase in this assayis an APP substrate and an APP derived product. The APP derived productcan be detected by various methods such as those described above, butfluorescent detection methods are preferred. The PAGE in gel activityassay can also be used to detect proteases other than secretases usingsuitable substrates. The in gel activity assay may also use othersuitable gels, such as, for example, agarose. In contrast to the PAGE ingel protein staining assays described below, the PAGE in gel activityassay determines only those proteins having protease activity ratherthan all proteins.

[0145] In a native PAGE in gel activity assay, the sample is in asolution which preserves protein complexes composed of proteinsassociated together by non-covalent and covalent bonds in their “native”state. Thus, a native PAGE in gel activity assay can determine theproteolytic activity of a protein complex. If a protein complex has suchactivity, that complex is referred to as a “protease complex.” Aprotease complex is two or more proteins associated together by anon-covalent bond, such as, for example, an ionic bond, or a non-peptidecovalent bond, such as, for example, a disulfide bond, and at least oneof the proteins has protease activity. A β-secretase protease complex isa protease complex that cleaves an APP substrate.

[0146] Referring to FIG. 10, another PAGE method that the Mono Sfraction was subjected is the “non-reducing SDS-PAGE in gel activityassay.” Like the native PAGE in gel activity assay, the non-reducingSDS-PAGE in gel activity assay also determined the β-secretaseendoprotease activity of the Mono S fraction in the PAGE gel. But thisassay differs in that it contains the detergent SDS, hence the term“SDS-PAGE.” SDS separates proteins associated together by a non-covalentbond. A “non-reducing in gel assay” means that the assay does notcontain a reducing agent, such as, for example, β-mercaptoethanol. Suchreducing agents sever covalent disulfide bonds between and withinproteins. Thus, in the non-reducing SDS-PAGE in gel activity assay,proteins associated by a non-covalent bond are separated from each otherbut those proteins that are linked by a disulfide bond are not.

[0147] The substrate used in all in gel activity assays was the peptideZ*Phe-Arg-MCA (PENINSULA LABORATORIES, San Carlos, Calif.;). ThePhe-Arg-MCA sequence of that sequence mimics the Val-Lys-Met sequence inthe β-secretase recognition site because both contain a hydrophobicamino acid adjacent to a positively charged amino acid and the MCAgroup, as discussed above, mimics a P1′ amino acid. As such, cleavage ofthe Arg-MCA bond in the Z*Phe-Arg-MCA substrate is equivalent tocleaving the Lys-Met bond in the β-secretase recognition site or in theZ*Val-Lys-Met-MCA substrate. That later substrate was not used for thein gel assay because, as discussed above, an aminopeptidase is requiredto detect cleavage of that substrate by Peak I.

[0148] Native PAGE in gel activity assays were conducted as follows. TheZ*Phe-Arg-MCA substrate was embedded into the gel by copolymerization ofZ*Phe-Arg-MCA (250 μ) with resolving gel (8 7×0.1 cm, NOVEX gelcassette, San Diego, Calif.;) components consisting of 12%polyacrylamide with 0.16% bis-acrylamide and 0.375 Tris-HCl, pH 8.8. Thestacking gel was 6% polyacrylamide, 0.16% bis-acrylamide, and 0.125 MTris-HCl, pH 6.8, prepared according to Laemmli (Laemmli, U. K. Nature227:259, 680-685 (1970)). The Mono S fraction (2-4 μl) was prepared innative sample buffer containing 50 mM Tris-HCl, pH 8.3, and 2% glycerol,and electrophoresed in the gel at 4° C. in a running buffer consistingof 25 mM Tris-HCl, 192 mM glycine, pH 8.3 for 2.5 hours at a constantcurrent of 25 mAmp. The gel was then washed in cold 2.5% Triton X-100solution for 10 minutes, and with cold sterile water for 10 minutes.β-secretase endoprotease cleavage of the substrate Z*Phe-Arg-MCAembedded in the gel was conducted by incubating the gel at 37° C. for 2hours in 100 mM citric acid-NaOH, pH 5.0, 1 mM EDTA, 1 mM DTT, and 10 mMCHAPS. AMC fluorescence in the gel was visualized under a UVtransilluminator. The fluorescent image was photographed with KodakDC120 digital camera, and analyzed with the EDAS120 image softwaresystem, which allows quantitative image analysis.

[0149] The native PAGE in gel activity assay of the Peak I Mono Sfraction resulted in a wide broad band of faint fluorescence. Thatresult is characteristic of a protease complex and shows that theactivity in Peak I is due to a protease complex. Moreover, the resultshows that the protease complex cleaves the Arg-MCA bond because thatcleavage must occur for fluorescence to be detected and fluorescence wasdetected without an aminopeptidase being present. Since the Arg-MCA bondin the Z*Phe-Arg-MCA substrate is equivalent to the Lys-Met bond in theβ-secretase recognition site, the protease complex also cleaves theLys-Met bond in that substrate.

[0150] The non-reducing SDS-PAGE in gel activity assay was conducted asdescribed for the native PAGE in gel activity assay, except that thestacking and resolving gels contained 0.1% SDS, the sample buffercontained 1.5% SDS, and the electrophoresis was conducted for 1.5 hours.The non-reducing SDS-PAGE in gel activity assay showed 3 distinct,precise and intense fluorescent bands corresponding to proteins havingmolecular weights of approximately 88, 81, and 66 kDa. The 3 proteinscleaved the Arg-MCA bond in the Z*Phe-Arg-MCA substrate becausefluorescence was produced without aminopeptidase. Moreover, thoseproteins also cleave the Lys-Met bond in the β-secretase recognitionsite for the reasons discussed above.

[0151] The Peak I Mono S fraction was also subjected to “preparativenative PAGE.” This electrophoresis method was used to further isolatethe β-secretases. Native conditions using the MiniPrep Cell system(BIORAD, Richmond, Calif.). Tube gels (7 mm internal diameter) wereprepared with the resolving gel (10 cm) consisting of 6% polyacrylamide(with 0.16% bis-acrylamide and 0.375 M Tris-HCl, pH 8.8) and a stackinggel (1 cm) of 4% polacrylamide (with 0.11% bis-acrylamide and 0.125 MTris-HCl, pH 6.8), prepared according to the manufacturer's protocol.The Mono S fraction (200 to 300 μl) in native sample buffer containing25 mM Tris-HCl, 192 mM glycine, pH 8.3, and 10% glycerol was subjectedto electrophoresis in the native tube gel at a constant power of 1 wattat 4° C. for 48 hours in running buffer consisting of 25 mM Tris-HCl,192 mM glycine, and pH 8.3. During electrophoresis, fractions (0.6ml/fraction) were eluted in running buffer at a flow rate of 0.02ml/minute; stability of eluted β-secretase endoprotease activity wasimproved with adjustment of fractions to pH 6.0 using an equal volume of0.1 M citric acid-NaOH, pH 4.5. Fractions were immediately assayed forZ*Val-Lys-Met-MCA cleavage in the presence of aminopeptidase M, or forZ-Phe-Arg-MCA without aminopeptidase M as described (Azaryan, A. V. andHook, V. Y. H., FEBS Lett. 341, 197-202 (1994)). After preparativenative gel electrophoresis, one peak of β-secretase endoproteaseactivity was observed for cleavage of the substrate Z*Val-Lys-Met-MCA.

[0152] The preparative native PAGE sample containing the activity wasfurther analyzed by various PAGE methods, including the non-reducingSDS-PAGE in gel activity assay described above. That assay resulted inthe same 3 activity bands having molecular weights of about 88, 81, and61 kDa obtained from the Mono S fraction run in that assay.

[0153] The preparative native PAGE sample was also analyzed in anon-reducing SDS-PAGE in gel protein staining assay which detects theproteins present in the gel. In contrast to the in gel activity assay,the protein staining assay detects all proteins present in a sufficientamount to be detected without regard to protease activity. Thenon-reducing SDS-PAGE in gel protein staining assay was conducted in asimilar manner as the activity assay, but was silver stained to identifythe proteins and resulted in 3 definite and precise bands correspondingto proteins having molecular weights of about 88, 81, and 36 kDa.

[0154] The results obtained from the non-reducing SDS-PAGE in gelprotein staining and activity assays were compared. The 88 and 81 kDaproteins observed by silver staining correlated with the two β-secretaseendoproteolytic activities at those weights in the activity assay. Butno protein was detected in the protein staining assay corresponding tothe 61 kDa activity band. This result implied that the amount of proteinat that position may have been insufficient to be detect by silverstaining. If that is the case, the 61 kDa protein had a very highspecific activity because intense activity was observed at thatposition. No activity was detected in the activity assay at the positioncorresponding to the 36 kDa protein, indicating that the 36 kDa proteindoes not have β-secretase endoproteolytic activity.

[0155] The preparative native PAGE sample was further analyzed in areducing SDS-PAGE in gel protein staining assay. Like the staining assaydescribed above, this assay also detected the proteins present in thegel without regard to proteolytic activity. But since this assay wasconducted in the presence of a reducing agent, β-mercaptoethanol,disulfide bonds were severed. The assay was run as described above forthe protein staining assay except that the gel and sample buffercontained β-mercaptoethanol. Four proteins having molecular weights ofapproximately 66, 60, 33, and 29 kDa were detected.

[0156] The reducing SDS-PAGE in gel protein staining assay resulted inmore and on average proteins of lower molecular weight than did thecorresponding non-reducing assay. That difference indicates that thepreparative native PAGE sample contained proteins having disulfide bondswhich were severed by the reducing agent to produce a larger number ofproteins with lower molecular weights. In particular, the 88 and 81 kDaproteins had such bonds severed because only lighter proteins wereobserved under reducing conditions. The 33 and 36 kDa proteins obtainedunder reducing and non-reducing conditions may be the same proteinbecause their weights are similar.

[0157] The results obtained from the reducing SDS-PAGE in gel proteinstaining and the non-reducing SDS-PAGE in gel activity assays werecompared. The 88 and 81 kDa proteins having activities contained one ormore disulfide bonds that were severed under the reducing conditions.The 60 kDa and 61 kDa proteins in silver staining and activity assayswere about the same weight and may be the same protein.

Isolation of β-SECRETASES From Peak II

[0158] The procedure used to isolate Peak II-A and Peak II-B from PeakII is diagramed in FIG. 11. The Sephacryl S200 fractions containing PeakII were pooled and further purified using Mono Q ion exchange FPLCchromatography (referred to as “Mono Q FPLC”). The fraction that did notbind to that column contained Peak II β-secretase endoprotease activity(referred to as the “unbound Peak II” or “Peak II-A”). The fraction thatbound to the column was eluted using a NaCl gradient from zero to 0.5 MNaCl, and also contained Peak II β-secretase endoprotease activity(referred to as “bound Peak II” or the “Peak II-B”). Peak II-B wasfurther purified by a second Mono Q column, with elution of theβ-secretase activity by a pH gradient of pH 7.0 to pH 4.0 generated bypolybuffer 74 (PHARMACIA, Piscataway, N.J.), performed as describedpreviously (Krieger, T. K. and Hook, V. Y. H., ibid.). Since Mono Q FPLCis an anion exchange chromatography, the unbound Peak II is a proteinthat is less electronegative than the Peak II-B protein.

EXAMPLE XIII β-secretase Endoprotease Activities Obtained DuringIsolation of β-secretases

[0159] The total (relative fluorescence units/0.5 hr) and specific(relative fluorescence units/mg protein) of the β-secretase endoproteaseactivity without and with aminopeptidase M (−APM, +APM, respectively)was determined for fractions obtained in the isolation proceduredescribed in Example XII. All assays were conducted as described inExample IX. The activities obtained are summarized in Table III. TABLEIII TOTAL ACTIVITY SPECIFIC ACTIVITY ISOLATION STEP −APM +APM −APM +APMLysate 11 12 1.8 1.9 Soluble extract 12 12 2.6 2.5 Membrane 0.4 0.6 1.72.3 Con-A bound^(a) 19 75 367 1.5 × 10³ Con-A unbound^(b) 8 9 2 2 Peak ISephacryl S200 13 275 2.0 × 10³ 4.2 × 10⁴ CF fraction 38 496 3.0 × 10³3.8 × 10⁴ Mono S fraction 16 300 5.0 × 10⁵ 9.3 × 10⁶ Prep. SDS-PAGEND^(c) 30 1.0 × 10⁷ 2.0 × 10⁷ Peak II Sephacryl S200 63 75 6.0 × 10⁴ 7.2× 10⁴ Mono Q FPLC 15 16 5.5 × 10⁵ 6.0 × 10⁵ Peak II-A Mono Q FPLC 6 63.1 × 10⁴ 4.4 × 10⁴ Peak II-B

[0160] The total activity of the lysate and the soluble extract withoutaminopeptidase M was about 92% and 100% of that with the aminopeptidase,respectively, and thus were aminopeptidase insensitive. The solubleextract contained about 100% of the total activity in the lysate, butthe membrane pellet contained only about 4% of that activity, indicatingthat the β-secretase endoprotease activity is not bound to thechromaffin vesicle membranes.

[0161] The eluted Con-A bound fraction assayed without and withaminopeptidase M had about 158% and 625% of the total activity in thelysate, respectively. The increase in the total activity indicated thatan inhibitor or competitive substrate, such as APP protein, may beremoved at this step. The eluted Con-A bound fraction had a totalactivity that was somewhat aminopeptidase sensitive as the activitywithout aminopeptidase M was approximately 25% of that with theaminopeptidase.

[0162] The Con-A unbound fraction contained the endogenous β-secretaseaminopeptidase activity which was not present in the eluted Con-A boundfraction. As such, Peak I and Peak II subsequently purified from theeluted Con-A bound fraction did not contain significant endogenousaminopeptidase activity.

[0163] Peak I from the Sephacryl S200 isolation step was highlyaminopeptidase sensitive, having a total activity of only about 4.7%without aminopeptidase M as and with the aminopeptidase. Moreover, PeakI assayed with the aminopeptidase had about 367% and 2292% of the totalactivity in the eluted Con-A bound fraction and lysate, respectively,again indicating possible removal of an inhibitor or competitivesubstrate.

[0164] Continuing with the isolation of Peak I, the CF fraction also wasaminopeptidase sensitive as the total activity without aminopeptidase Mwas about 7.6% of that with the aminopeptidase. Again the total activitywas increased, this time by about 180% and 4,133% of that from theSephacryl S200 fraction and the lysate, respectively, as measured withaminopeptidase M and again raising the possibility that an inhibitor orcompetitive substrate was removed.

[0165] The Mono S fraction of Peak I remained very aminopeptidasesensitive, having a total activity without aminopeptidase M of about5.3% of that with the aminopeptidase. But the total activity of the MonoS fraction was about 60% and 2,500% of that in the CF fraction andlysate, respectively. This indicates that the Mono S isolation step maylose some activity but that the activity remains well above that in thelysate.

[0166] The preparative SDS-PAGE isolation of Peak I resulted in 10% and250% of the activity in the Mono S fraction and lysate, respectively.Moreover, the activity after this step, unlike the previous isolationsteps, became quite unstable indicating that the preparative SDS-PAGEisolation step may remove an activator or stabilizing agent.

[0167] Returning to the isolation of Peak II by Sephacryl S200, the PeakII had about 27% of the activity of Peak I. In other words, Peak I hadabout 3 times more β-secretase endoprotease activity than did Peak II.But Peak II was relatively aminopeptidase insensitive as the totalactivity without aminopeptidase M was about 84% of that with theaminopeptidase. Peak II total activity assayed with aminopeptidase M wasthe same as that in the eluted Con-A bound fraction indicating that thisisolation step does not remove an inhibitor, an APP substrate, anactivator, or a stabilizing agent.

[0168] After Mono Q FPLC isolation, Peak II-A and Peak II-B were foundto be aminopeptidase insensitive. The combined total activity of PeakII-A and Peak II-B was about 32% of the total activity in the SephacrylS200 fraction with aminopeptidase M. Peak II-A and Peak II-B had a totalactivity of about 133% and 66% of that in the lysate, respectively.

[0169] The specific activity showed that a very high degree of isolationwas obtained. Specifically, the preparative SDS-PAGE electrophoresisisolation step of Peak I resulted in about a 0.5×10⁶ and 1.0×10⁶purification from the chromaffin vesicle lysate as analyzed without andwith aminopeptidase, respectively. The Mono Q FPLC isolation of PeakII-A resulted in a 2.3×10⁵ and 3×10⁵ purification from the chromaffinvesicle lysate as analyzed without and with aminopeptidase,respectively. The Mono Q FPLC isolation step of the Peak II-B resultedin a 1.5×10⁴ and 2,2×10⁴purification from the chromaffin vesicle lysateas analyzed without and with aminopeptidase, respectively.

EXAMPLE XIV Protease Inhibitors of β-secretase Endoprotease Activity inPeaks I and II

[0170] The effect of various protease inhibitors on β-secretaseendoprotease activity in Peaks I and II was determined by the methoddescribed in Example XI. The results were expressed as a percentinhibition of the control (no inhibitor) is summarized in Table IV.TABLE IV PROTEASE Peak I Peak II CLASS INHIBITOR (Concentration) (%) (%)Control None 100 100 Cysteine E64c (10 μM) 0 0 Cysteine pHMB (1 mM) 6768 Serine PMSF (100 μM) 90 112 Serine Chymostatin (10 μM) 0 35 AspartylPepstatin A (100 μM) 85 132 Metallo EDTA (1 mM) 99 138 Metallo EGTA (1mM) 108 142 Metallo 1,10 Phenanthroline (500 μM) 31 72 NonspecificLeupeptin (100 μM) 0 0

[0171] Peak I and Peak II activities were maximally inhibited by thenonspecific protease class inhibitor leupeptin, the cysteine classinhibitor E64c, and the serine protease class inhibitor chymostatin. Theother cysteine class inhibitor, pHMB, slightly inhibited bothactivities. The other serine protease class inhibitor, PMSF, did notsignificantly inhibit either activity. The metallo protease classinhibitor 1,10 phenanthroline significantly inhibited Peak I, but onlyslightly inhibited Peak II. The other metallo protease class inhibitorsand the aspartyl protease class inhibitor pepstatin A did notsignificantly inhibit either activity.

[0172] Peak I and Peak II activities were identically inhibited by thecysteine protease class and nonspecific protease class inhibitors. Theserine, aspartyl and metallo protease classes inhibitors tended toinhibit Peak I activity more than Peak II.

[0173] The inhibition of Peak I and Peak II activities was compared withthat obtained for the chromaffin vesicle lysate (Example XI). All 3activities were completely inhibited by the cysteine protease classinhibitor E64c and the nonspecific protease class inhibitor leupeptin.The serine protease class inhibitor chymostatin and the cysteineprotease class inhibitor pHMB inhibited all activities, although thePeak I and Peak II activity was inhibited less than that of the lysate.The serine protease class inhibitor PMSF significantly inhibited thelysate activity but only slightly inhibited the Peak I and Peak IIactivities. The aspartic protease class inhibitor pepstatin A slightlyinhibited the lysate and Peak I activities but increased the activity ofPeak II. Except for the 1,10 phenanthroline, none of the metallo classprotease class inhibitors inhibited any activity and, in some cases,increased the activity.

EXAMPLE XV Confirmation of Cleavage Specificities of the Peak I, PeakII-A, and Peak II-B β-Secretase Endoprotease Activities

[0174] As discussed above, the substrates Z*Val-Lys-Met-MCA andZ*Phe-Arg-MCA mimic the β-secretase recognition site in the APP protein.The fluorescent MCA that resulted from the cleavage of those substratesestablished the cleavage specificities of the Peak I, Peak II-A, andPeak II-B β-secretases. In particular, those results showed that themajority of the endoprotease activity in Peak I cleaved the Lys-Met bondamino terminally adjacent to the β-secretase scissile bond in theβ-secretase recognition site of the APP protein. Those results alsoshowed that the majority of the endoprotease activity in Peak II-A andPeak II-B cleaved the β-secretase scissile bond in the β-secretaserecognition site of the APP protein.

[0175] To confirm the Peak I cleavage specificity, electrospray massspectrometry (EMS) was also used to analyze the APP derived productsresulting from the cleavage of the Z*Val-Lys-Met-MCA substrate by thePeak I activity. The cleavage assay was conduced by the method describedin Example XII without aminopeptidase M. The APP derived products werethen analyzed by a commercial EMS facility (SCRIPPS RESEARCH INSTITUTE,La Jolla, Calif.). The EMS analysis confirmed that the Peak I activitycleaved the Lys-Met bond in the Z*Val-Lys-Met-MCA substrate.

[0176] To confirm the cleavage specificities of the Peak I, Peak II-A,Peak II-B activities, another APP substrate was reacted with each ofthose activities and the APP derived products analyzed by EMS. The APPsubstrate Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe (SEQ ID NO.:5) containsthe 5 amino terminal and 4 carboxyl terminal amino acids to theβ-secretase scissile bond in the APP protein. The substrate wascommercially produced and purified to greater 95% purity by standardreverse phase high pressure liquid chromatography methods. The cleavageassay of Example XII was used without the aminopeptidase M and withoutthe Z*Val-Lys-Met-MCA substrate, but with theSer-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe (SEQ ID NO.:5) substrate (14μg/assay). The APP derived products were then subjected to a C8 reversephase high pressure liquid chromatography, eluted with an acetonitrilegradient in 0.1% TFA (trifluoroacetic acid), the peptides identified byabsorbance spectroscopy at 210-215 nm and collected (see Krieger T. K.and Hook V. Y. H., ibid. and Krieger et al., J. Neurochem. 59, 26-31(1992)). The EMS data of the eluted APP derived products confirmed thatthe majority of Peak I activity cleaved the Lys-Met bond and that themajority of the Peaks II-A and II-B activities cleaved the Met-Asp bond.

[0177] The above-identified references are expressly incorporatedherein. Although the invention has been described with reference to theexamples provided above, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the claims.

I claim:
 1. A substantially pure β-secretase comprising a protein havinga molecular weight selected from the group consisting of about 61, 81and 88 kiloDaltons (kDa) as determined by cleavage of an APP substratein a non-reducing SDS-PAGE in gel activity assay.
 2. The β-secretase ofclaim 1, wherein the protein cleaves the APP substrate at a β-secretaserecognition site.
 3. The β-secretase of claim 2, wherein the proteincleaves the β-secretase recognition site at a bond selected from thegroup consisting of Lys-Met and Val-Lys.
 4. The β-secretase of claim 3,wherein the protein cleaves the Lys-Met bond.
 5. A method of selectingan agent that inhibits a cleavage of an APP substrate comprisingcontacting the agent with the β-secretase of claim 1 and selecting theagent that inhibits the cleavage of the APP substrate by theβ-secretase.
 6. A method of inhibiting production of an Aβ peptide by acell comprising contacting the cell with the agent selected by themethod of claim 5 and thereby inhibiting production of the Aβ peptide bythe cell.
 7. A method of inhibiting production of an Aβ peptide by anAlzheimer's disease patient comprising administering to the patient theagent selected by the method of claim 5 and thereby inhibitingproduction of the Aβ peptide by the Alzheimer's disease patient.
 8. Asubstantially pure protease complex having a molecular weight betweenabout 180 and 200 kDa as determined by Sephacryl chromatography thatcleaves an APP substrate.
 9. The protease complex of claim 8, whereinthe protease complex cleaves the APP substrate at a β-secretaserecognition site.
 10. The protease complex of claim 9, wherein theprotease complex cleaves the β-secretase recognition site at a bondselected from the group consisting of Lys-Met and Val-Lys.
 11. Theprotease complex of claim 10, wherein the protease complex cleaves theLys-Met bond.
 12. The protease complex of claim 8, further comprisingproteins having a molecular weight selected from the group consisting ofabout 66, 60, 33 and 29 kDa as determined by a reducing SDS-PAGE in gelprotein staining assay.
 13. The protease complex of claim 8, furthercomprising proteins having a molecular weight selected from the groupconsisting of about 61, 81 and 88 kDa as determined by cleavage of anAPP substrate in a non-reducing SDS-PAGE in gel activity assay.
 14. Amethod of selecting an agent that inhibits a cleavage of an APPsubstrate comprising contacting the agent with the protease complex ofclaim 8 and selecting the agent that inhibits the cleavage of the APPsubstrate by the protease complex.
 15. A method of inhibiting productionof an Aβ peptide by a cell comprising contacting the cell with the agentselected by the method of claim 14 and thereby inhibiting production ofthe Aβ peptide by the cell.
 16. A method of inhibiting production of anAβ peptide by an Alzheimer's disease patient comprising administering tothe patient the agent selected by the method of claim 14 and therebyinhibiting production of the Aβ peptide by the Alzheimer's diseasepatient.
 17. A substantially pure β-secretase having a molecular weightbetween about 50 and 90 kDA as determined by Sephacryl chromatographyand that cleaves an APP substrate.
 18. The β-secretase of claim 17,wherein the protease complex cleaves the APP substrate at a β-secretaserecognition site.
 19. The β-secretase of claim 18, wherein theβ-secretase cleaves the β-secretase recognition site at a bond selectedfrom the group consisting of Met-Asp, Lys-Met and Val-Lys.
 20. Theβ-secretase of claim 19, wherein the β-secretase cleaves the Met-Aspbond.
 21. The β-secretase of claim 17, further comprising 2 proteinshaving different electronegative charges as determined by ion exchangechromatography.
 22. A method of selecting an agent that inhibits acleavage of an APP substrate comprising contacting the agent with theβ-secretase of claim 17 and selecting the agent that inhibits thecleavage of the APP substrate by the β-secretase.
 23. A method ofinhibiting production of an Aβ peptide by a cell comprising contactingthe cell with the agent selected by the method of claim 22 and therebyinhibiting production of the Aβ peptide by the cell.
 24. A method ofinhibiting production of an Aβ peptide by an Alzheimer's disease patientcomprising administering to the patient the agent selected by the methodof claim 22 and thereby inhibiting production of the Aβ peptide by theAlzheimer's disease patient.